Internal spike

Biological Samples. There were three types of biological samples obtained from workers at the plant urine, whole blood, and feces. All urine and blood samples were internally "spiked" at the factory with 1 yg/mL of a nitrosopiperidine (NPiP) standard. NPiP was used for spiking because it has a similar stability and recovery characteristic to nitrosomorpholine, and to provide a means of gauging the accuracy of the analytical methods. Due to the inability to perform homogeneous mixing on-site, the feces samples were not spiked until they were thawed upon return to the laboratory. Ethyl acetate extracts of urine samples were examined for the presence of N-nitrosodiethanolamine (NDEIA), a metabolite of NMOR, by HPLC-TEA. All samples were immediately frozen at the plant (-80°C) and kept at this temperature until analysis. 

Analysis for NDEIA Ten mL of the thawed urine were placed on another Preptube, pre-wet with ethyl acetate. The sample was washed with 60 additional milliliters of ethyl acetate and the effluent dried (rotary evaporator) to 1 milliliter. It was then analyzed by HPLC-TEA using a yNH2 column eluted with isooctane dichloromethane methanol (60 30 7). Recoveries for NDEIA using this method were approximately 70%. The percent recovery was judged by an internal spike of N-nitrosodipropanolamine. 

Immediately prior to analysis, 100 jiL of a 10 ppm In solution were added to a 10 mL aliquot of each sample as an internal spike to correct for instrument drift. Analyses were performed on a Fisons PQ 2 Plus quadmpole ICP mass spectrometer. Rather than produce calibration curves for each of the 41 elements analyzed, simple blank subtraction was used and the results for the artifacts were compared directly against geological source samples prepared and analyzed in the same way (55). 

A sixth spectrophotometric method for the quantitative determination of Pb + levels in blood uses CQ+ as an internal standard. A standard containing 1.75 ppb Pb + and 2.25 ppb CQ+ yields a ratio of Sa/Sis of 2.37. A sample of blood is spiked with the same concentration of Cu +, giving a signal ratio of 1.80. Determine the concentration of Pb + in the sample of blood. 

Caffeine in coffee, tea, and soda is determined by a solid-phase microextraction using an uncoated silica fiber, followed by a GC analysis using a capillary SPB-5 column with an MS detector. Standard solutions are spiked with G3 caffeine as an internal standard. 

Radiochemical methods of analysis take advantage of the decay of radioactive isotopes. A direct measurement of the rate at which a radioactive isotope decays may be used to determine its concentration in a sample. For analytes that are not naturally radioactive, neutron activation often can be used to induce radioactivity. Isotope dilution, in which a radioactively labeled form of an analyte is spiked into the sample, can be used as an internal standard for quantitative work. 

The most useful methods for quality assessment are those that are coordinated by the laboratory and that provide the analyst with immediate feedback about the system s state of statistical control. Internal methods of quality assessment included in this section are the analysis of duplicate samples, the analysis of blanks, the analysis of standard samples, and spike recoveries. 

Environment Internal Entering water temperature 85°F (29 C), exiting water temperature 115°F (46°C), pressure -120 psig (0.82 MPa), pH 7.5-7.6, gaseous chlorination 0.3-0.6 ppm free with spike of 3 ppm weekly 14-16 ppm orthophosphate 3.0-3.5 ppm zinc dispersant. External Cracked gasoline, 130 psi (0.9 MPa)... 

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 11.5 Chromatograms of plasma samples obtained with fully automated on-line SPE-LC (a) dmg-ffee human plasma (b) human plasma spiked with omeprazole (100 ng/ml) and phenacetin (internal standard 1000 ng/ml). Reprinted from Journal of Pharmaceutical and Biomedical Analysis, 21, G. Garcia-Encina et al., Validation of an automated liquid chromatograpliic method for omeprazole in human plasma using on-line solid-phase exti action, pp. 371 - 382, copyright 1999, with permission from Elsevier Science.

Figure 5.67 Reconstructed ion chromatograms for Idoxifene and internal standard (ds-Idoxifene using LC-ToF-MS for (a) double-blank human plasma extract, (b) extract of blank human plasma containing internal standard (IS), and (c) control-blank human plasma spiked with Idoxifene at 5 gml , the LOQ of the method. Reprinted from 7. Chromatogr., B, 757, Comparison between liquid chromatography-time-of-flight mass spectrometry and selected-reaction monitoring liquid chromatography-mass spectrometry for quantitative determination of Idoxifene in human plasma , Zhang, H. and Henion, J., 151-159, Copyright (2001), with permission from Elsevier Science.

Radioactivity Analysis. Samples of urine, feces, and tissues were combusted to COo and analyzed for radioactivity (5). By using this method the recovery of radioactivity from samples spiked with C was 95 dt 5%. To determine the radioactivity expired as CO2, 5-ml aliquots of the solution used to trap the CO2 were added to 15 ml of a scintillation counting solution containing 4 grams 2,5-diphenyloxazole (PPO) and 0.1 grams l,4-bis-2(5-phenyloxazolyl)-benzene (POPOP) per liter of 1 1 toluene 2-methoxyethanol. Samples were counted for radioactivity in a Nuclear Chicago Mark II liquid scintillation counter. Counting eflSciency was corrected by the internal standard technique. 

To validate the analytical procedure recovery experiments are performed. To this end, the CRM is spiked with a known mass of the analytes at a variety of concentration levels (at least three different levels) and the concentrations measured are compared to the expected concentrations in at least three separate experiments. The extraction step has been shown to be a critical step in the analytical procedure and it may be responsible for poor recoveries. The efficiency of this step can be assessed either by repetitive extraction of the sample or by the addition of internal standards prior to the extraction step with the assumption that the latter actually represent the behavior of the analytes of interest. 

All values of the IRMM Isotopic Reference Materials are traceable to the SI (the international system of base quantities and base units). Isotopic measurement results corrected by means of these Isotope Ratio Reference Materials have reduced (ISO/BIPM) uncertainties. Isotopic measruements carried out against these Spike Reference Materials are traceable to the SI, if carried out properly. Further details are available from IRMM website see Chapter 8. 

For precise measurement of isotopic composition by mass spectrometry, it is also common to use either a natural, known isotopic ratio to correct for instrumental mass fractionation (e g., internal normalization) or to add a tracer for this purpose. For example for natural uranium samples, one can use the natural U/ U of 137.88 to correct for fractionation. Alternatively, one can use an added double spike of ratio -unity... 

Also, a chromatographic profile or fingerprint of trace unknowns can be established and monitored, so that if product performance unexpectedly changes, there will be a starting point for troubleshooting. The effects of experimental variables on sample recoveries should be measured directly by controlled variation of an experimental factor, using the reference standard, or suitable external standards, or spiked addition of an external standard to the reference standard. A detailed example of the use of internal and external standards is presented in Chapter 4. 

Method development for high-pressure ashing and closed microwave digestion was reported for wet oxidation and extraction of Pb, Cd, Cr and Hg from various food packaging materials [57]. Use of HPA resulted in the highest median recoveries of the spiked elements (Pb and Cd, 92% Cr, 97% Hg, 83%). The use of In as an internal standard improved the accuracy... 

FIGURE 14.2 Achiral-chiral separation of S-Ketorolac (S-l), R-Ketorolac (R-l), p-hydro-xyketorolac enantiomers (2,3) and internal standard Naproxen (4). (a) Urine blank (b) blank urine spiked with 1.5pg/mL each of racemic ketorolac and p-hydroxyketorolac (c) Nondeconjugated urine sample taken from a patient who received 10 mg of oral racemic ketorolac. Reprinted from Diaz-Perez et al. (1994) with permission from John Wiley Sons. 

In the bioanalytical studies, the basic calibration should be prepared in the same biological matrix as in the samples of the intended study, which can be achieved by spiking the matrix with known concentration of the analyte. In this case, a blank sample, a zero sample (blank and internal standard), six to eight nonzero samples covering the expected range (including the anticipated QL) should be evaluated as part of the linearity study [27]. 

Olafsson [478] has reported on the results obtained in an international intercalibration for mercury in seawater. Sixteen countries participated in this exercise, which involved analysis of a seawater and seawater spiked with 15.4 and 143 ng/1 mercury. The results show good accuracy and precision in the recovery of mercury for the majority of calibrations, but serious errors in the low-level determinations on the seawater. 

In the analysis of seawater, isotope dilution mass spectrometry offers a more accurate and precise determination than is potentially available with other conventional techniques such as flameless AAS or ASV. Instead of using external standards measured in separate experiments, an internal standard, which is an isotopically enriched form of the same element, is added to the sample. Hence, only a ratio of the spike to the common element need be measured. The quantitative recovery necessary for the flameless atomic absorption and ASV techniques is not critical to the isotope dilution approach. This factor can become quite variable in the extraction of trace metals from the salt-laden matrix of seawater. Yield may be isotopically determined by the same experiment or by the addition of a second isotopic spike after the extraction has been completed. 

Some methods may involve a procedure known as standard additions. This is when the internal chemical standard is identical to the analyte and a known amount of it is added to a sample solution. Clearly, if the internal chemical standard is the same chemical as the analyte, then in order to determine the analyte level in the sample, it will be necessary to measure the sample twice, i.e. once without any chemical standard added and once with the standard added. There are several ways of carrying out the process of standard additions two are described here. The addition of a chemical standard which is the same as the analyte is also called spiking . 

Sample preconcentration was performed by means of an automated on-line SPE sample processor Prospekt-2 (Spark Holland, Emmen, The Netherlands). Oasis HLB cartridges (Waters, Barcelona, Spain) were used to preconcentrate cannabi-noids present in the water samples whereas isolation of the rest of the compounds was done in PLRPs cartridges (Spark Holland). Before extraction, influent samples were diluted with HPLC water (1 9, v/v) to reduce matrix interferences and to fit some analyte concentrations, e.g., cocaine (CO) and benzoylecgonine (BE), within the linear calibration range. A sample volume of 5 mL was spiked with the internal standard mixture (at 20 ng/L) in order to correct for potential losses during the analytical procedure, as well as for matrix effects. Elution of the analytes to the LC system was done with the chromatographic mobile phase. 

---