Calibration surrogate internal standard

Calibration standards can be of two types external standards and internal standards. With external standards, multiple concentrations of the standards are injected, areas are measured, and a calibration curve is platted. Unknown samples are then injected, chromatograms run, and areas are calculated and compared with the calibration curves to determine amounts of each compound present. With internal standards, known amounts of an internal standard are added to each known concentration of standard compound and areas or peak height response factors relative to those of the internal standard are calculated. When unknowns are run, a known amount of internal standard is added to the unknown sample, response factors are calculated relative to the internal standards, and amounts of each unknown present are calculated from the standards calibration factors. Internal standards are usually used to correct for variations in injection size due to different operators and injection techniques. Internal standards can also be used to correct for extraction variation in GC/MS target compound quantitation, this standard is referred to as a surrogate standard. Generally, an internal standard is used for one purpose or the other, not both at the same time. 

This section provides a simplified introduction to the methods by which calibration solutions containing analytical (or reference) standards, with or without internal standards, are used in practice to measure amounts of analytes in unknown samples. The main deficiency of this brief account is its lack of any attempt to take into account experimental uncertainties (both random and systematic) and thus the level of confidence in the results thus obtained. While this book is directed towards analyses in which mass spectrometry is used as the chromatographic detection technique, most of the following discussion is applicable also to other detectors the main exception concerns use of isotope-labeled surrogate internal standards, for which only mass spectrometry can provide adequate detection. A much more complete account of this material, including a discussion of the associated random and systematic errors, is given in Section 8.5. 

Knownfixed amounts of a surrogate internal standard (Qsis O are spiked into a series of calibration solutions (varying concentrations Cf = Qa"/V") and also into the analytical sample that contains the unknown amount of analyte (Qf). 

The two cases discussed thus far are obviously not best practice examples but such circumstances may arise on occasion also, the preceding discussion allowed demonstration of the benefits of matrix matched cahbrators over clean solutions of the analytical standard. In situations where neither a surrogate internal standard nor suitable blank matrix are available, the Method of Standard Additions (MSA) is a preferred analytical method it is included in this sub-category for convenience, although strictly speaking it involves a non-zero intercept for the experimental line that serves both as calibration and measurement. In this method the analytical sample itself is used as a kind of blank into which the calibration standard is spiked. As will become apparent, linearity of response is a prerequisite for accurate results and this implies that multiple experiments must be performed at... 

The need for a surrogate internal standard (SIS) and the ways in which it is used in practice were discussed in Sections 2.6.3 and 8.5.2. Questions concerning choice and availability of an SIS were introduced in Section 2.2.3 and Table 2.1, and are discussed further below. All of the concerns about chemical and chiral purity, applicable to the analytical standard (Section 9.4.4), apply also to the SIS however, the absolute chemical purity is not as crucial for the SIS in cases where both calibration and measurement are performed using response ratios of analyte SIS where the absolute quantity of SIS detected does not enter into the final determination (Section 8.5.2b, essentially the signal from the SIS is used as a normalizing factor for the analyte responses). In the few instances where a volumetric internal standard is required (Sections 2.2.4 and 8.5.2a) the only concern is that the VIS is stable and provides no interferences (direct or indirect) that co-elute with the analyte. 

Continuing calibration for a Series Method is performed using calibration check compounds. Surrogate compounds are added to the matrix before sample preparation to evaluate recovery levels. To check GC retention times, internal standards are added to a sample after its preparation for analysis. 

Quality Assurance/Quality Control. QA/QC measures included field blanks, solvent blanks, method blanks, matrix spikes, and surrogates. Percent recovery was determined using three surrogate compounds (nitrobenzene-d5, 2-fluorobiphenyl, d-terphenyl-diQ and matrix spikes (naphthalene, pyrene, benzo[ghi]perylene) the recoveries ranged from 80 to 102%. Separate calibration models were built for each of the 16 PAHs using internal standards (naphthalene-dg, phenanthrene-dio, perylene-di2). Validation was performed using a contaminated river sediment (SRM 1944) obtained from NIST (Gaithersburg, MD) accuracy was <20% for each of the 16 analytes. 

After a sample sequence has been analyzed, the analyst evaluates a computer printout with the raw analytical data and the computer-calculated analytical results. The analyst verifies that the QC acceptance criteria for daily calibration verifications, the surrogate and internal standards, and laboratory QC samples have been met. This review enables the analyst to determine whether reanalysis of samples will be required. Sample dilutions may be also needed if analyte concentrations in samples exceed the instrument calibration range. 

The modem GC data system will produce a report of peaks detected with the retention time, peak area, and peak height. In order to identify the analytes of interest and quantify the data, a series of calibration standards are required to be analyzed followed by samples. The calibration standards will identify retention times for analytes, surrogates, and internal standards. With the exception of MS analysis, compounds are identified in chromatograms based solely on their retention time. Positive confirmation can be done by analyzing the same sample extract on a different type (polarity) of GC column. If the compound is detected at the same concentration from both GC columns, then the data can be reported (e.g., US EPA Method 8081—OC Pesticides—requires analysis on a DB-5 column with confirmatory analysis on a DB-17 column). For MS analysis, multiple ion chromatograms... 

Quantitative data of selected target compounds were obtained by integration of specific ion chromatograms extracted from the TIC. Injection volume and sample volume inaccuracies were corrected for by using internal standard compounds as a surrogate standard. An external four-point-calibration generated from reference compounds was used for quantification. 

The calibration technique of standard addition is often used to minimize matrix effects in the quantification of organotins. External calibration is also used by many groups, howeveg the use of an internal standard is highly recommended with the nature of the best compound to use dependent on the measurement system involved. Tripropyltin chloride has been used by many groups as a surrogate to monitor the whole method process, and species such as tetra-butyltin and tetrapentyltin have been used as internal standards added just prior to GC analysis. 

The internal standards that are used are acenaphthene-surrogate standards are 1,3-dimethy 1-2-nitrobenzene, triphenylphosphate, and perylene-calibration standards in ethyl acetate over the range from 0.1 to 10 p,g/L are injected. A longer splitless time (2 min), which increases the amount of analyte loaded unto the column, and the thinner-fihn column... 

Diphenylphthalate, diphenylisophthalate, and dibenzylphthalate are the surrogate standards that are added at 50 p,g/L to water samples and at 830 fig/Kg to soils prior to extraction. An internal standard or external standard calibration can... 

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