Quantification method validation

Chandra, A., Rana, J., and Li, Y., Separation, identification, quantification, and method validation of anthocyanins in botanical supplement raw materials by HPLC and HPLC-MS, J. Agric. Food Chem., 49, 3515, 2001. 

Today, when a pesticide with no detectable residues is registered for use, a Tolerance or maximum residue limit (MRL) is established at the lowest concentration level at which the method was validated. However, for risk assessment purposes it would be wrong to use this number in calculating the risk posed to humans by exposure to the pesticide from the consumption of the food product. This would be assuming that the amount of the pesticide present in all food products treated with the pesticide and for which no detectable residues were found is just less than the lowest level of method validation (LLMV). The assumption is wrong, but there is no better way of performing a risk assessment calculation unless the limit of detection (LOD) and limit of quantification (LOQ) of the method were clearly defined in a uniformly acceptable manner. 

Thanks to the efforts of a continuously increasing number of research groups, CE has by now been accepted as a highly efficient separation technique for qualitative purposes, with about 1500 CE-related documents appearing annually in analytical journals. However, CE has not yet been fully established as a quantification method, mainly due to the predominance of HPLC techniques applied in standard, validated analytical protocols. 

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. 

The purpose of an analytical method is the deliverance of a qualitative and/or quantitative result with an acceptable uncertainty level. Therefore, theoretically, validation boils down to measuring uncertainty . In practice, method validation is done by evaluating a series of method performance characteristics, such as precision, trueness, selectivity/specificity, linearity, operating range, recovery, LOD, limit of quantification (LOQ), sensitivity, ruggedness/robustness, and applicability. Calibration and traceability have been mentioned also as performance characteristics of a method [2, 4]. To these performance parameters, MU can be added, although MU is a key indicator for both fitness for purpose of a method and constant reliability of analytical results achieved in a laboratory (IQC). MU is a comprehensive parameter covering all sources of error and thus more than method validation alone. 

Limit of detection (LOD) sounds like a term that is easily defined and measured. It presumably is the smallest concentration of analyte that can be determined to be actually present, even if the quantification has large uncertainty. The problem is the need to balance false positives (concluding the analyte is present, when it is not) and false negatives (concluding the analyte is absent, when it is really present). The International Union of Pure and Applied Chemistry (IUPAC) and ISO both shy away from the words limit of detection, arguing that this term implies a clearly defined cutoff above which the analyte is measured and below which it is not. The IUPAC and ISO prefer minimum detectable (true) value and minimum detectable value of the net state variable, which in analytical chemistry would become minimum detectable net concentration. Note that the LOD will depend on the matrix and therefore must be validated for any matrices likely to be encountered in the use of the method. These will, of course, be described in the method validation document. 

The fundamental parameters for bioanalytical validations include accuracy, precision, selectivity, sensitivity, reproducibility, stability of the drug in the matrix under study storage conditions, range, recovery, and response function (see Section 8.2.1). These parameters are also applicable to microbiological and ligand-binding assays. However, these assays possess some unique characteristics that should be considered during method validation, such as selectivity and quantification issues. 

The assay has been validated and the results of validation demonstrate that the standard curve is linear over the concentration range of 100-2000 ng/mL. The assay is reproducible and accurate, with recovery of the analyte and internal standard in the range of 80-90 %. The analysis requires 0.5 mL of plasma and has a limit of quantification of 70 ng/mL. The stability of plasma samples stored at -20 °C has been demonstrated for up to 12 weeks. Autoinjector stability has been demonstrated for over 13 h and freeze-thaw stability has been demonstrated for 3 freeze-thaw cycles. The procedure has a sample throughput of at least 30 specimens per day. The assay meets the guidelines for bioanalytical methods validation for human studies (Shah et al. 1991). 

There are no detailed recommendations for analytical procedures in the field of biotechnological production of drugs, in contrast to the recommendations made by the FDA for bioanalytical methods [16], The absence of detailed guidelines for analytical method validation in this field is surprising. It is important to validate the quantification of both substrate and product during the process, at definite time intervals, to ensure proper calculation of the kinetics of the process i.e., the coefficients of substrate conversion, and production rates must be adequately calculated. The aim of the study in pa-... 

We have applied this protocol to the evaluation of the measurement uncertainty for a method for the determination of three markers (Cl solvent red 24, Cl solvent yellow 124 and quinizarin (1,4-dihydroxyanthra-quinone)) in road fuel. The method requires the extraction of the markers from the sample matrix by solid phase extraction, followed by quantification by HPLC with diode array detection. The uncertainty evaluation involved four experimental studies which were also required as part of the method validation. The studies were precision, trueness (evaluated via the analysis of spiked samples) and ruggedness tests of the extraction and HPLC stages. The experiments and uncertainty calculations are described in detail in Part 2. A summary of the uncertainty budget for the method is presented in Fig. 3. 

Ion-selective electrodes (ISEs) represent the current primary methodology in the quantification of S-Li [11-13], Moreover, ISE modules are parts of large and fully automated clinical chemistry analysers. In practice, the validation parameters are most often chosen in terms of judging the acceptability of the new measurement system for daily use. For this reason, the first approach was to study whether the detected imprecision fulfilled the desired analytical quality specifications. Secondly, proficiency testing (PT) results from past samples were of great value in predicting future bias. The identity of the three ISE methods was evaluated using patient samples. The analytical performance was checked after 6 months routine use. Without any exception, method validations always mean an extra economical burden. Therefore, the validation parameters chosen and processed have to be considered carefully. 

None of the lipoprotein methods described in this section has been used widely enough to have been validated in independent studies to the same extent as have been the Friedewald and beta-quantification methods. In most cases, the identities of the lipoprotein contributing to the LDL cholesterol measurement have not been adequately established. Further evaluations should better define the relationships between these new methods and current reference and routine methods. 

Method validation makes use of a series of tests to determine its performance characteristics and to establish the method s acceptance for general use. The following are a list of criteria associated with the validation of a method selectivity and specificity, linearity and calibration, accuracy or trueness, range, precision, limit of detection, limit of quantification, ruggedness and application. 

To overcome this inherent problem of SAE signal quantification, we developed a new quantification method called SPAQ, which is based on a defined overlap between consecutive ultrasound images. The SPAQ technology is schematically described in Fig. 7.6, and has been validated in agarose phantoms and ex-vivo in animal tissue [23, 24]. 

Each lot of unprocessed bulk drug derived from mammalian cell culture (e.g., the harvesf) should be assessed for bioburden, and shown to be free of adventitious vimses and mycoplasma as described in ICH Q5A. Titers of endogenous retrovims (e.g., type C particles) in chnical lots and at least the first three consistency lots should be quantified for comparison to the validated clearance capacity of the purification process [5.] Generally, the quantification of retrovirus in the unprocessed hulk drug is carried out using transmission electron microscopy (TEM), though quantification by validated real time PCR methods is also acceptable [5, 37]. 

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