Alternative reference methods, validation

Collaborative studies are also aimed at the validation of alternative reference methods. Comparative tests of various analytical or operating procedures can be used to validate a method of choice or even to establish a close correlation among a method involving... 

HPLC methods can usually be transferred without many modifications, since most commercially available HPLC instruments behave similarly. This is certainly true when the columns applied have a similar selectivity. One adaptation, sometimes needed, concerns the gradient profiles, because of different instrumental or pump dead-volumes. However, larger differences exist between CE instruments, e.g., in hydrodynamic injection procedures, in minimum capillary lengths, in capillary distances to the detector, in cooling mechanisms, and in the injected sample volumes. This makes CE method transfers more difficult. Since robustness tests are performed to avoid transfer problems, these tests seem even more important for CE method validation, than for HPLC method validation. However, in the literature, a robustness test only rarely is included in the validation process of a CE method, and usually only linearity, precision, accuracy, specificity, range, and/or limits of detection and quantification are evaluated. Robustness tests are described in references 20 and 59-92. Given the instrumental transfer problems for CE methods, a robustness test guaranteeing to some extent a successful transfer should include besides the instrument on which the method was developed at least one alternative instrument. 

Similar conclusions were recently reported both by Hodgeson et al. and by Paur. These findings provide further validation of the ultraviolet method for calibrating air monitoring instruments and establish gas-phase titration as an alternative primary reference method. 

In order to estimate these indicators, it is necessary to use various analytical methods, generally approved by USEPA or standardized by others national or international organizations such as APHA (American Public Health Association) ASTM, AOAC-Intemational or ISO. In addition to reference methods, alternative procedures or new methods can be tested and approved, according to EPA guidelines or APHA, ASTM, AOAC-intemational, and ISO protocols (AOAC, 1999 APHA, 1998 ASTM, 1999 ISO, 2001). In order to approve and validate new test procedures, confirm laboratory performance and update approved methods (Table 1.2.4) USEPA publishes both approved test methods and procedures and those that have not been promulgated. 

Certified reference materials should be used in the validation process to establish the capability of the method and then as part of on-going quality control procedures to ensure that the capability of the method is maintained in routine operation. Alternatively, reference or in-house materials (which are often less expensive) can be used in on-going quality control procedures, provided the materials have been evaluated against the certified reference material. 

A validation plan should be written before the initiation of the prestudy validation experiments. Alternatively, reference to an appropriate SOP can be made to ensure that a documented outline exists for the experiments required for prestudy validation. This plan can be a stand-alone document or can be contained in a laboratory notebook or some comparable format. The documentation should include a description of the intended use of the method under consideration and a summary of the performance parameters to be validated that should include, but may not be limited to, standard curve, precision and accuracy, range of quantification, specificity and selectivity, stability, dilutional linearity, robustness, batch size, and run acceptance criteria. The plan should include a summary of the proposed experiments and the target acceptance criteria for each performance parameter studied. 

If new analogues of public health significance are discovered, they should be included in the analysis. Standards must be available before chemical analysis is possible. Total toxicity shall be calculated using conversion factors based on the toxicity data available for each toxin. The performance characteristics of these methods shall be defined after validation following an internationally agreed protocol. Biological methods shall be replaced by alternative detection methods as soon as reference materials for detecting the toxins are readily available, and the methods have been validated [72]. 

Over the past few years, the LC-MS and LC-MS/MS techniques have been successfully applied to the quantification and identification of YTXs. Several drafts for methods have already been published, which would facilitate immediate replacement of the mouse bioassay. However, Decision 2002/225/EC (Annex) requires validation of alternative methods by use of reference materials for all the individual compounds specified. So, the lack of pure reference samples of most YTXs prevents the substitution of the mouse bioassay as the routine or reference method for years to come. 

Developments of quality assurance of the analytical speciation procedures and results are of prime importance at this point. Validation of new methodologies should be carried out if possible with certified reference materials (CRMs) (with a matrix as similar as possible to the real sample certified for the sought species). Unfortunately, very few CRMs are commercially available today for chemical speciation. Thus, alternative approaches for validation using complementary methods based on different mechanisms of separation and complementary detectors are now in order. 

When the assumption of error-free x-values is not valid, either in method comparisons or, in a conventional calibration analysis, because the standards are unreliable (this problem sometimes arises with solid reference materials), an alternative comparison method is available. This technique is known as the functional relationship by maximum likelihood (FREML) method, and seeks to minimize and estimate both x- and y-dlrection errors. (The conventional least squares approach can be regarded as a special and simple case of FREML.) FREML involves an iterative numerical calculation, but a macro for Minitab now offers this facility (see Bibliography), and provides standard errors for the slope and intercept of the calculated line. The method is reversible (i.e. in a method comparison it does not matter which method is plotted on the x-axis and which on the y-axis), and can also be used in weighted regression calculations (see Section 5.10). 

Similar to quantitative methods, qualitative tests should also undergo a method validation. The measuring system of a qualitative test usually transforms a quantitative result into a negative or positive report or in some cases a semi-quantitative outcome however, the absolute numerical concentration of the analyte itself is not reported. Qualitative tests can be validated by using a series of samples with known concentrations of analyte that fall either side of the positive-negative cut-off. These known values may be assigned by an alternative method, or may be reference material. It is particularly important to assess reproducibility of results around a concentration of the analyte of interest that is clinically important such as around a diagnostic cut-off. A method comparison study can also be undertaken with a comparator. The new method can usually be implemented when predefined criteria are fulfilled. 

This validation typically requires samples with radiolabeled analytes. However, alternative approaches are proposed which involve (i) comparison with extraction of samples using a procedure which has been previously validated rigorously, (ii) comparison with extraction of samples by a very different technique or (iii) analysis of a certified reference material. Generally, this validation should be performed with samples containing analyte incurred by the route by which residues would normally be expected to arise. The simplest option (i) requires fully validated and documented enforcement methods provided by the manufacturer of a pesticide. 

For complex sample types, if there is any doubt concerning the ability of the method to unambiguously identify and measure the analyte of interest, check the method using a closely matched matrix reference material, or check the sample using an alternative validated method. 

Like ANNs, SVMs can be useful in cases where the x-y relationships are highly nonlinear and poorly nnderstood. There are several optimization parameters that need to be optimized, including the severity of the cost penalty , the threshold fit error, and the nature of the nonlinear kernel. However, if one takes care to optimize these parameters by cross-validation (Section 12.4.3) or similar methods, the susceptibility to overfitting is not as great as for ANNs. Furthermore, the deployment of SVMs is relatively simpler than for other nonlinear modeling alternatives (such as local regression, ANNs, nonlinear variants of PLS) because the model can be expressed completely in terms of a relatively low number of support vectors. More details regarding SVMs can be obtained from several references [70-74]. 

Oscillation of the dried granulation and lubricant was accomplished in every instance using a no. 10 wire screen. Reference to the no. 12 screen, the alternative method for pulverizing the batch, must be deleted from the manufacturing instructions for the process to be validated retrospectively. 

The validity of any comparative study is dependent on the reliability of the identification of the samples in the study. Not all researchers are experts in the field of identification of samples, nor do all researchers have quick and ready access to expert systemetists who can accomplish the task of identification. The importance of verification of sample identity for comparative studies is vital. We describe several methods by which researchers can obtain and identify samples from the wild, and we suggest methods by which voucher samples can be obtained for future reference to these collected samples. We outline alternatives to collection of samples from the wild, such as purchase from stock centers and biological supply companies. Museum collections can also be extremely helpful in obtaining complete organismal samples for comparative studies. 

---