Sample preparation spiking procedures

This sample preparation involved, firstly, an extraction and the elimination of the solid matrix by filtration and, secondly, a concentration procedure employing a solid phase extraction cartridge. The compounds of interest were separated solely by dispersive interactions with the reversed phase. In the example given, the corn meal was spiked with the aflatoxins. 

To investigate the effect of both factors (i.e., sample preparation and detector sensitivity), solutions of different concentrations near the ICH reporting limits are prepared by spiking known amounts of related substances into excipients. Each solution is prepared according to the procedure and analyzed repeatedly to determine the S/N ratio. The average S/N ratio from all analyses at each concentration level is used to calculate the QL or DL. The following equation can be used to estimate the QL at each concentration level. Since different concentration levels give different QLs, typically the worst-case QL will be reported as the QL of the method. 

Intrinsic Accuracy. Intrinsic accuracy indicates the bias caused by sample matrix and sample preparation. In this approach, a stock solution is prepared by using known quantities of related substance and drug substance. The stock solution is further diluted to obtained solutions of lower concentrations. These solutions are used to generate linearity results. In addition, these linearity solutions of different concentrations are spiked into placebo. The spiked solutions are prepared according to the procedure for sample analysis. The resulting solutions, prepared from the spiked solution, are then analyzed. If the same stock solution is used for both linearity and accuracy and all of these solutions are analyzed on the same HPLC run, the response of linearity (without spike into matrix) and accuracy (with spike into matrix) can be compared directly. Any differences in response indicate the bias caused by matrix interference or sample preparation. To determine the intrinsic accuracy at each concentration level, one can compare the peak area of accuracy (with matrix) with that of linearity (without matrix) at the same concentration (Figure 3.11). This is the simplest approach, and one would expect close to 100% accuracy at all concentration levels. 

Molybdenum isotope ratio measurements by MC-ICP-MS (Plasma 54) have been carried out using Zr or Ru elemental spikes to study the mass discrimination during the whole analytical procedure including sample preparation.146 A laboratory fractionation of Mo isotopes of about 0.15 % is observed during ion exchange by offline Mo separation. Using this analytical technique, possible natural isotope variation of Mo can be determined with a precision of 0.02 %. 

Since the spiking-based approach also measures the effectiveness of the sample preparation procedure, care should be taken to mimic the actual sample preparation as closely as possible. It should be always considered that in many cases the analyte added to a sample may not behave in the same manner as the same analyte biologically incurred at relatively high concentrations, and particularly with methods involving a large number of sample preparation steps, percentage recoveries may be lower. 

One of the most important trends to simplify these complications is the generation of simple, rapid, and reliable procedures for sample preparation. Method development and setup require the use of materials of known compositions, for example, certified reference materials. Therefore, spiking experiments have to be performed for method quality control. Under such experimental conditions, emphasis has to be placed on the spiking procedures as they exert an influence on the recovery values. Although present scientific knowledge is not perfect, the use of spiking experiments helps to minimize the errors. The integration and automation of all steps between sample preparation... 

It is important to point out that the quality of analytical results is not immediate it can only be achieved if an extensive set of measures are adopted and complied with. Therefore, in parallel to the development of the QA concept, QC systems were introduced as an important tool supporting the QA of chemical measurements. The QC process of examination of laboratory performance in time should always follow QA. QC thus comprises a set of operational techniques and activities used to check whether the requirements for quality are fulfilled. In practice, QC in an analytical chemistry laboratory implies operations carried out daily during the collection, preparation, and analysis of samples, which are designed to ensure that the laboratory can provide accurate and precise results. QC procedures are intended to ensure the quality of results for specific samples or batches of samples and include the analysis of reference materials (RMs), blind samples, blanks, spiked samples, duplicate, and other control samples.2... 

Lewisite 1 was used for spiking in the sixth and in the ninth proficiency test. The participants that failed to identify lewisite 1 did not describe an effective lewisite 1 sample preparation procedure (12) in their analysis report, which could indicate that no such procedure had been employed. The participants performances in the identification of lewisite 1 are summarized in Table 3. 

Methylphosphonic acid was used for spiking in the fifth and in the tenth proficiency test. Many participants, in particular, failed to identify methylphosphonic acid in the tenth test for reasons that are difficult to trace from their analysis reports. It can only be speculated that the high concentration of salt ( %% NaCl) in the sample has been the cause most of these participants did not describe an effective sample preparation procedure (e.g. cation exchange) for the removal of salts, and employed GC/MS as an indirect analysis technique. Indirect GC/MS analysis of methylphosphonic acid requires derivatiza-tion salts are known to influence the derivatization reaction negatively. The participants performances in the identification of methylphosphonic acid are summarized in Table 6. 

The soil sample was problematic in the second trial proficiency test. Only six of the 15 participating laboratories successfully identified both N,N-diethylaminoethanol (CAS 100-37-8) and A-ethyl-diethanolamine (EDEA, CAS 139-87-7). For these particular spiking chemicals, efficient TEA/methanol extraction followed by a silylation procedure was essential in the sample preparation. One laboratory missed both spiking chemicals, probably because TEA/methanol extraction was not carried out. Another laboratory also missed them but for a different reason. Instead of TEA, ethyldimethy-lamine (EDMA, CAS 598-56-1) was used as an extractant, and although this should not have had a dramatic effect on the recoveries of the spiking chemicals, since EDMA should be as good a modifier as TEA, the laboratory then derivatized the 1 % EDMA/methanol extract with BSTFA, thus removing any chance of success. Chemicals in alcohol solutions cannot be silylated, and this must... 

The recovery has been assessed for this assay in the following way 5 replicates of all three QC levels have been prepared according to the sample preparation recipe, which is given in this assay. The resulting data are compared to blank brain extracts (again 5 replicates), which were spiked with the analyte to the expected concentration level after the extraction procedure. In this way, the completeness of the extraction process can be determined. 

The recovery experiment carried out for this assay resembles somehow the sum of two potential effects The loss of compound during the sample preparation process (in this case on-line extraction) and a potential matrix effect during sample analysis. However, since an online sample clean up procedure is used here, these two potential effects cannot be separated in the usual way (One experiment would be the comparison of a processed spiked plasma sample with a blank plasma sample spiked post sample processing in order to determine the recovery of the sample preparation step. In a second experiment, again a processed blank plasma sample would be spiked post processing and the result would be compared with the response of a spiked solvent sample in order to reveal a potential matrix effect during analysis). 

The internal standard (I.S.) method is a more accurate method. The I.S. technique can compensate for both instrumental and sample preparation errors and variations (e.g., dilution and extraction) [45, 46], Sample pretreatment steps such as extraction often result in sample losses, and a proper I.S. standard should be chosen to mimic the variations in these steps. Thus, both the accuracy and precision of quantitative data increase if an I.S. is included in the procedure. The I.S. should be similar but not identical to the analyte, and the two should be well resolved in the chromatographic step. The standard curves are obtained from standards of blank samples spiked with different known concentrations of the analyte of interest and addition of an I.S. at constant concentration. Also to the unknown samples the same constant concentration of the I.S. is added. The standard samples are processed in parallel with the unknown samples. In the calibration curve, the ratios of analyte to I.S. peak area (or height) are plotted versus the concentration of the analyte. A proper I.S. in a bioanalytical chromatographic method should fulfill the following requirements [44] ... 

High recovery of the analyte(s) from the matrix is a desirable outcome of sample preparation, and is therefore an important characteristic of the extraction procedure. The absolute recovery is the ratio of the response measured for a spiked sample (in matrix) treated according to the whole analytical procedure to that of a non-biological sample spiked (in aqueous solution) with the same quantity of the analyte substance and directly injected into the chromatographic system [81], The relative recovery is the ratio of the responses between extracted spiked samples (in matrix) and extracted spiked pure samples (in aqueous solution). The relative recovery can be used, together with the absolute recovery, to reveal whether sample losses in the extraction are due to matrix effects or to bad extraction. 

The choice of which method to use may be decided by the type of polymer/plastic material and the metal required for analysis. With the analysis of a completely unknown sample it may be necessary to carry out trial and error tests of different methods with and without spiking before accepting the final sample preparation method. Once confidence is achieved in the method of analysis, the procedure is noted for future reference. 

The recovery function and rate can be determined for each individual step of sample preparation. This allows the specihc improvement of the critical steps. Afterwards the same procedure can be performed with spiked samples for the determination of matrix effects. 

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