Spike recovery tests

Referee Laboratories and Spike Recovery Testing. Outside laboratories, with demonstrated performance records, can be used to evaluate the suitability of a candidate method when none of the other accuracy testing options is feasible. However, This technique provides a very weak form of accuracy assessment. Indeed, it provides a comparability check, not an accuracy measure. Similarly, spike recovery tests provide only weak evidence of method accuracy. Quantitative spike recovery only indicates that the added form of the analyte was recovered. If the added form responds differently toward sample preparation or detection the utility of spike recovery testing remains doubtful. 

Accuracy. In the quantitative method that is used to measure the heavy metal quantity in the drug substance, the accuracy is usually represented by the recovery rate obtained from a spiked recovery test where lead is added to the samples. Since the heavy metals limit test specified in monograph specifications is a test where the intensity of coloring of the samples with sodium sulfide is compared with that of the control solution, it is necessary to confirm that heavy metal components can be detected fully in the process of test solution preparation. The Heavy Metals Limit Test in JP specifies four preparation methods for the test solutions. An appropriate method will be selected and used for further testing. The test method that gives the best recovery rate is to be adopted. The procedure is as follows ... 

The spiked recovery test cannot produce a favorable result. The following precautions should be taken at the time of preparation of the test and control solutions from samples. 

The diligent analyst would develop a robust method with rigorous matrix effect tests on multiple lots, including hemolyzed and lipidemic samples. An initial test would be a spike-recovery evaluation on at least six individual lots. Samples should be spiked at or near the LLOQ, and at a high level near the ULOQ. If matrix interference were indicated by unacceptable relative error (RE) percentage in certain lots, the spiked sample of the unacceptable lots should be diluted with the standard calibrator matrix to estimate the minimum dilution requirement (MDR) at and above which the spike-recovery is acceptable. The spike-recovery test should then be repeated with the test samples diluted at the MDR. Note that this approach will increase the LLOQ for a less sensitive assay. If sensitivity is an issue, then other venues will be required to address the matrix effect problem. For example, the method can be modified to include sample clean-up, antibodies and/or assay conditions may be changed, or the study purpose may be tolerable to acknowledge that the method may not be selective for a few patients (whose data may require special interpretation). 

A procedure for the determination of molybdenum in serum, red blood cells, and urine is described. The low concentration of molybdenum in most unexposed individuals requires the sensitivity obtained using atomic absorption spectrophotometry and electrothermal atomization. Spike recovery tests indicate that low temperature ashing is required for accuracy. Severe matrix interferences preclude wet ashing or high-temperature ashing as sample pretreatments. Using the method described, it is possible to distinguish between industrially exposed and unexposed individuals. 

In spike-recovery tests, samples from individual lots of matrix (>3 lots for exploratory and >10 for advanced studies) are spiked with concentrations of the reference standard from the lower and middle (upper) portions of the curve, assayed, and compared to the corresponding neat (unmodified or unspiked) samples. Spike recovery is calculated by subtracting calculated concentration in the unspiked samples... 

FIGURE 6.6 Spike recovery test of urine samples from normal and patients for NTx. 

The relevance of the requested spike recovery test, practical aspects of the spiking [e.g. slurry (possible over-estimation of recovery) or dry down (better admixture but possible degradation risk)] and the problems of degradation of calibrants were discussed and will be the subjects of further studies. Apparent... 

Quality control (QC) is as important in speciation analyses as it is in other areas of analysis. While there are a growing number of certified reference materials available that contain specific forms of an element, for example, tributyltin, methylmercury, this is the exception rather than the rule. Instead, other standard QC approaches need to be used. These include the use of spike-recovery tests and internal standards, comparisons with the results from other methods. 

Accurate analysis was achieved using optimum LC-MS/MS conditions LOD was 0.58 ng/ml or higher and LOQ was 2.5 ng/ml in all the analytes. The spike recovery test was conducted with internal standards and the average recovery was 97.9% or higher [relative standard deviation (RSD) 5.8%, n = 6], thereby suggesting that this method is useful to quantitatively determine PFCs in house and carpet dust. This method revealed that PFC concentration in house dust was extremely high compared to that in environmental samples, including environmental water and air. 

Method validation is needed to demonstrate the acceptability of the analytical method. A recovery test on a chemical being determined should be performed in order to verify the reliability of the series of analyses. Recovery studies are usually conducted by spiking untreated sediment with the target chemical at the deteetion limit, quantitation limit and in the range of 10-50 times the detection limit. The method is considered acceptable when the recoveries typically are greater than 70%. When the recovery is less than 70%, an improvement in the analytical methods is needed. Where this is not possible for technical reasons, then lower recovery levels may be acceptable provided that method validation has demonstrated that reproducible recoveries are obtained at a lower level of recovery. Analysis is usually done in duplicate or more, and the coefficient of variation (CV) should be less than 10% to ensure that recoveries will be consistently within the range 70-110%. 

Maintenance of the integrity of matrices used for tield fortification samples is of the utmost importance to the tield investigator since cross-contamination of the matrix prior to tield fortification could lead to tield spike recoveries for matrices of a questionable nature. The matrices to be used for tield fortification samples must be maintained in a pristine state prior to use in the tield. Inadvertent contamination of the tield fortitication matrices will invalidate any tield fortification samples which are prepared. Extreme care must be taken to ensure that these matrices stay free of any residue of the test substance. 

TESTING FOR SYSTEMATIC ERROR IN A METHOD COMPARISON TEST FOR A SET OF MEASUREMENTS VERSUS TRUE VALUE - SPIKED RECOVERY METHOD (COMPARE 71 WORKSHEET)... 

Here the concept of statistical control is not applicable. It is assumed, however, that the materials in the run are of a single type. Carry out duplicate analysis on all of the test materials. Carry out spiking or recovery tests or use a formulated control material, with an appropriate number of insertions (see above), and with different concentrations of analyte if appropriate. Carry out blank determinations. As no control limits are available, compare the bias and precision with fitness-for-purpose limits or other established criteria. 

Precision and accuracy Quantitative analysis by NMR is very precise with relative standard deviations for independent measurements usually much lower than 5%. The largest errors in NMR measurements are likely due to sample preparation, not the NMR method itself. If a good set of standards is available and all NMR measurements for the test and standard samples are performed under the same acquisition conditions, the quantitative results can be readily reproduced on different instruments operated by different analysts at different times. Therefore, good intermediate precision can also be achieved. An accurate quantitative NMR assay will require accurately prepared standards. The accuracy of an NMR assay can be assessed, for example, by measuring an independently prepared standard or an accurate reference sample with the assay. In many cases, a spike recovery experiment can also be used to demonstrate the accuracy of an NMR assay. 

The accuracy of an analysis can be determined by several procedures. One common method is to analyze a known sample, such as a standard solution or a quality control check standard solution that may be available commercially, or a laboratory-prepared standard solution made from a neat compound, and to compare the test results with the true values (values expected theoretically). Such samples must be subjected to all analytical steps, including sample extraction, digestion, or concentration, similar to regular samples. Alternatively, accuracy may be estimated from the recovery of a known standard solution spiked or added into the sample in which a known amount of the same substance that is to be tested is added to an aliquot of the sample, usually as a solution, prior to the analysis. The concentration of the analyte in the spiked solution of the sample is then measured. The percent spike recovery is then calculated. A correction for the bias in the analytical procedure can then be made, based on the percent spike recovery. However, in most routine analysis such bias correction is not required. Percent spike recovery may then be calculated as follows ... 

The accuracy of a method is defined as the closeness of the value obtained to known or accepted values. Accuracy can be determined in a number of ways, depending on the nature of the CZE method and availability of orthogonal techniques to compare results. If practical, spike recovery studies (i.e., testing to determine whether recovery matches the amount of a known analyte or impurity spiked) are good alternatives to orthogonal assay comparisons. ICH guidelines also allow method accuracy to be inferred, once specificity, linearity, and precision are established. 

Accuracy Recovery Linearity Spiked placebo test Intercept of a linearity plot... 

Another performance requirement often specified is spike recovery. Sometimes, response to analyte is affected by something else in the sample. We use the word matrix to refer to everything else in the sample other than analyte. A spike, also called a fortification, is a known quantity of analyte added to a sample to test whether the response to a sample is the same as that expected from a calibration curve. Spiked samples are analyzed in the same manner as unknowns. For example, if drinking water is found to contain 10.0 pg/L of nitrate, a spike of 5.0 pg/L could be added. Ideally, the concentration in the spiked portion found by analysis will be 15.0 pg/L. If a number other than 15.0 pg/L is found, then the matrix could be interfering with the analysis. 

Thus, control charts measure both the precision and accuracy of the test method. A control chart is prepared by spiking a known amount of the analyte of interest into 4 to 6 portions of reagent grade water. The recoveries are measured and the average recovery and standard deviation are calculated. In routine analysis, one sample in a batch is spiked with a known concentration of a standard and the percent spike recovery is measured. An average of 10 to 20 such recoveries are calculated and the standard deviation about this mean value is determined. The spike recoveries are plotted against the frequency of analysis or the number of days. A typical control chart is shown below in Figure 1.2.2. 

Limits or combination of limits which, when exceeded, trigger analyst intervention. These limits may be defined statistically or based on test method requirements. Control limits may be assigned to method blanks, check standards, spike recoveries, duplicates and reference samples. Most control limits for toxicity tests are based on thrice the standard deviation of the mean (i.e., one in every 100 tests would be expected to exceed the control limits due to chance alone). Volume 1(10). 

Post digestion spike addition or recovery test... 

If the above approach creates practical problems, an alternative is to perform single analyses on a minimum of five test portions of the study sample. The standard deviation of replicate analysis results is an indicator of sample homogeneity and method precision. The disadvantage of this approach is that it does not provide a simultaneous measure of the analytical variance under the homogeneity test conditions. Analytical variance must be estimated from historical data (e.g. method validation) or spiked recoveries run with the homogeneity test samples. 

---