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Ruggedness testing in analytical chemistry
Abstract
Very high standards of accuracy and precision are currently expected from analytical methods. This is particularly true for pharmaceutical applications, due not only to the potential toxicity of drugs but also to the strict controls of regulatory bodies. This paper deals with the validation of methods by testing ruggedness to changes in the analytical method conditions. As a ruggedness test examines a number of variables simultaneously and hence a large experimental response surface, the design has to be carefully chosen. Advice is offered on the test design, including the selection of factors and the levels at which to test them. Some results are shown for high-performance liquid chromatography methods.
... The specificity of an analytical method may be defined as the ability to unequivocally determine the analyte in the presence of additional components such as impurities, degradation products, and matrix [34][35][36]. The specificity was evaluated by injecting the analytical placebo and it was found that the signal measured was caused only by the analyte. ...
... The LOD and LOQ were calculated using signal-to-noise ratio method [34][35][36]. LOD was taken as the concentration of the analyte where the signal-to-noise ratio was 3, and it was found to be 50 ng mL −1 . LOQ defined as the analyte concentration at a signal-to-noise ratio of 10 was found to be 200 ng mL −1 . ...
... These values indicate that the method is suitable for detection and quantification of OLP over a wide range of concentrations. [34][35][36]. Solutions of three different concentrations of OLP were analysed in seven replicates during the same day (intraday precision) and five consecutive days (interday precision). Within each series, every solution was injected in triplicate. ...
An accurate, precise, sensitive, and rapid isocratic reversed phase high-performance liquid chromatographic (RP-HPLC) method for the analysis of olanzapine (OLP) in bulk drug and in tablets has been developed and validated. Analysis was performed on a 150 mm × 4.6 mm, 5 μm particle Intersil ODS 3V column with 10 mM disodium hydrogen phosphate buffer (pH 7.4)-acetonitrile (35 : 65) (v/v) as mobile phase at a flow rate of 1.0 mL min−1 with UV detection at 254 nm; the constant column temperature was 40°C. The runtime under these chromatographic conditions was less than 8 min. The calibration plot was linear over the concentration range of 2.5–20.0 μg mL−1 with limits of detection and quantification values of 50 and 200 ng mL−1, respectively. The precision and accuracy of the method were assessed by determination of validation data for precision (intraday RSD values of 0.11–0.28%, interday RSD values of 0.15–0.46%), accuracy (0.87–2.80% intraday, 0.33–1.80% interday), and specificity, in accordance with the ICH guidelines. The stability of standard solution and tablet extract was also studied over a period of 24 h. The method was applied for the determination of OLP in tablets with satisfactory results.
... As recommended in the ICH guidelines and the Dutch Pharmacists guidelines, a robustness assessment was performed during the development of the analytical procedure [38]. The ruggedness [39] of the two methods was assessed by comparison of the intra-day and inter-day assay results for MIC, NYS, HCA and NS that were performed by two analysts. The CV% values for intra-day and inter-day assays of the four analytes in the Monicure NH vaginal suppositories performed in the same laboratory by two analysts did not exceed 3.8%, indicating the ruggedness of the two methods. ...
... Recovery data resulting from the proposed HPLC and HPTLC methods were statistically compared with those of the reported HPLC methods: [14,17,30,39] for MIC NYS, HCA and NS, respectively, using one way ANOVA. It was found that the calculated F values did not exceed the critical value of the Fratio at α = 0.05, indicating no significant differences between the proposed and reported methods (Table 7) ...
... The two proposed analytical methods were compared in term of sensitivity, precision and accuracy to further assess their applicability in the analysis of real samples. [14,17,30,39] for MIC NYS, HCA and NS, respectively. b The critical value of F-ratio is 3.89 at α = 0.05. ...
Two new validated methods for the quantification of miconazole (MIC), nystatin (NYS), hydrocortisone acetate (HCA) and neomycin (NS) by high performance thin layer chromatography (HPTLC)-densitometry and reverse-phase high-performance liquid chromatography procedure coupled with photodiode array detector (RP-HPLC-DAD) were developed and compared. HPTLC separations were performed using a mobile phase, ethylacetate:methanol:glacial acetic acid (60:40:0.4, v:v:v) for MIC, NYS, HCA and ethylacetate for NS. For HPLC-DAD analysis, using an isocratic elution system, separation of all compounds was achieved. Good resolution and quantization were achieved, accuracy and precision, as well as detection and quantitation limits of the two methods, were evaluated and compared. Excellent linearity was observed for all of the standard calibration curves, and the correlation coefficients were above 0.9997. HPTLC limits of quantitation were 15.37×10⁻²,9.8×10⁻²,13.32×10-2 and 15.19×10-2 µg/mLfor MIC, NYS, HCA and NS respectively, whereas HPLC limits were 6.80×10⁻², 6.56×10⁻²,1×10-1 and 10.16×10⁻² µg/band for MIC, NYS, HCA and NS respectively. In comparison with HPLC, HPTLC is less expensive and faster procedure, requiring 2-3 h to analyse 10-12 samples on a single plate. The developed methods were proved to be specific, robust and accurate for the determination of cited drugs in pharmaceutical preparations.
... Another aim of performing a robustness test can be to define SST limits. 9,12,13,[15][16][17] The SST limits are then determined based on the experimental data from a robustness test, while frequently they are chosen arbitrarily based on the experience of the analyst. ...
... In a robustness test, the following steps can be distinguished: 6,9,17,18 (1) selection of the factors to be evaluated and their levels, ...
... Usually, the factors in a robustness test are operational or environmental factors. 6,[9][10][11]17,19 The former are selected from the operating procedure of the method, while the latter are not necessarily specified in this procedure. Those factors, which are most likely to vary when a method is transferred between different laboratories, analysts or instruments, are selected. ...
In biomedical and pharmaceutical analysis, and particularly in the pharmaceutical industry, much attention is paid to the quality of the obtained analytical results because of the strict regulations set by regulatory bodies. Proper method validation demonstrates the fit of an analytical method for a given purpose. In this context, robustness testing has become increasingly important.The setup and treatment of results of such a robustness test are discussed in this chapter. All steps of the test are considered. Finally, a literature review and critique of applications of robustness testing of CE methods has been provided.
... ICH guidelines prescribe that the robustness of a method should be assessed during the development phase (or at the beginning of the validation), and not at the end of method validation [2]. Robustness can be evaluated by statistical experimental design to examine simultaneously the influence of the variation in several method variables, e.g., mobile phase flow rate, temperature, type of column, slope of the gradient, buffer pH, ionic strength, detector wavelength, additives type and concentration, etc., on the outcome (response) of a method [3][4][5]. Based on the objective, two strategies can be adapted for robustness studies. If the investigation only meant to verify that the already validated method is robust, screening designs such as Plackett-Burman design (PBD) [5][6][7], fractional factorial design [5,8,9] or supersaturated design is employed. ...
... Hence it is better to derive the system suitability limits from the results of the experimental design, as proposed by Mulholland et al. [3,21] who make use of the extreme results to define the SST limits. Since, these extreme results may not be the worst we propose to use the worstcase situations to define the SST limits. ...
A chromatographic method newly optimized to identify and assay four antihypertensive drugs in tablet dosage forms was complemented by a robustness test. The best system suitability criteria for numerous responses were evaluated on the basis of the robustness test results. Generally speaking, it is difficult to achieve a total satisfactory solution. Situations may also become ambiguous if the system suitability limits for few responses of a robust method are violated. In this context, it becomes crucial to redefine these limits based on the robustness test results. In the present study, the extreme experimental (worst-case) conditions that offer worst result but still acceptable and likely to occur were predicted from the robustness test effects. Eventually, replicated experiments were executed in such worst conditions and the system suitability test (SST) limits were determined.
... Ruggedness of the method was determined by measuring the absorbance of the standard solution (6µg/ml) by two different analysts and two different instruments [19]. The robustness was assessed by measuring the absorbance at two different wave lengths (278 and 282 nm). ...
The main objective is to develop and validate the UV-spectrophotometric method for the estimation of Tetrahydrocurcumin as per ICH guidelines. Materials and Methods: A simple, rapid, accurate, and economical UV-spectrophotometric method has been developed for the estimation of tetrahydrocurcumin. Results: Theλ max of tetrahydrocurcumin was found to be 280nm. The drug follows linearity in the concentration range 2-12 µg mL-1 with a correlation coefficient value of 0.9975. The accuracy of the method was checked by recovery experiment performed at three different levels, i.e., 50%,100% and 120%. The % recovery was found to be in the ranged between 97.81-101.39 with < 1 %RSD, thereby indicating the low variability and a strong agreement between experimental and true values. Ruggedness of the proposed method was studied with the help of two analysts. Conclusion: The above method was a rapid tool for routine analysis of tetrahydrocurcumin.
... Baseline experiments assess the performance of a method under assumed optimum testing conditions. Robustness experiments test the robustness of a method by identifying variables or testing conditions that might affect the method's results [20,55]. ...
Vector control interventions play a fundamental role in the control and elimination of vector-borne diseases. The evaluation of vector control products relies on bioassays, laboratory and semi-field tests using live insects to assess the product’s effectiveness. Bioassay method development requires a rigorous validation process to ensure that relevant methods are used to capture appropriate entomological endpoints which accurately and precisely describe likely efficacy against disease vectors as well as product characteristics within the manufacturing tolerance ranges for insecticide content specified by the World Health Organization. Currently, there are no standardized guidelines for bioassay method validation in vector control. This report presents a framework for bioassay validation that draws on accepted validation processes from the chemical and healthcare fields and which can be applied for evaluating bioassays and semi-field tests in vector control. The validation process has been categorized into four stages: preliminary development; feasibility experiments; internal validation, and external validation. A properly validated method combined with an appropriate experimental design and data analyses that account for both the variability of the method and the product is needed to generate reliable estimates of product efficacy to ensure that at-risk communities have timely access to safe and reliable vector control products.
Supplementary Information
The online version contains supplementary material available at 10.1186/s12936-023-04717-w.
... Hoy en día la robustez se conoce como la "medida de la capacidad de un método analítico de no ser afectado considerablemente por variaciones pequeñas pero deliberadas de los principales parámetros del método analítico" (Thompson, Ellison y Wood, 2002;USP 36, 2009). Por otro lado, el término solidez (en inglés ruggedness) se utiliza con frecuencia como sinónimo, sin embargo, este término se encuentra relacionado en la actualidad con el concepto de reproducibilidad que se encuentra en el VIM (Mulholland, 1988; van Leeuwen y col., 1991); desafortunadamente hoy en día, aún persisten confusiones en este parámetro, por lo cual, en ocasiones, se asocian estudios colaborativos a ensayos de robustez. ...
Aplicativo y guía disponibles en https://validar.inm.gov.co/validaR/
... Robustness.-The robustness in analytical methods measures the viability of a method without being affected by the slight variation of the optimal parameters that occur during measurements without affecting the results obtained from the method. 57,58 The robustness of the current method suggested by the response current wave stability was demonstrated with slight changes in the experiential condition. Here the studied parameters involved; the variation of pH (±0.2), equilibrium time in each voltammetric measure (10 ± 5 s). ...
In this work, a sensitive and selective voltammetric determination for Manidipine in pharmaceutical formulations and biological fluids have been studied based on the enhanced electrochemical response at surface nano-iron oxide modified screen-printed electrode, Scanning electron microscopy (SEM) and cyclic voltammetry (CV) were used to confirm the morphology, structure of the as-prepared nanospheres electrochemical characterizations different modified electrodes. The modified nano iron oxide modified printed electrode (FSPE) exhibits voltammetric with high sensitivity, stability, and its applicability over a wide range for the determination of MAN in 0.04 M universal buffer (pH = 4.00 ± 0.01), SWV method shows linearity over the concentration range 60–925 nM. LOD and LOQ are 8.5nM and 28.4 nM respectively. The statistical validation comparison with the reported technique was also performed for the determination of MAN in authentic and pharmaceutical formulations. Moreover, the modified nano-screen printing sensor showed fine selectivity, reproducibility, and stability, and it was successfully used for the simultaneous determination of MAN in the presence of ascorbic acid and uric acid in dentate urine samples with appropriate results.
... However, performing a robustness test late in the validation procedure involves the risk that when a method is found not to be robust, it should be redeveloped and optimized. One can save money and efforts spent atthat pointtherefore the performance of a robustness test has been shifting to earlier points of time in the life of the method [1][2][3][4][5]. According to USP general chapter <1225>, "the robustness of an analytical procedure is a measure 215 | P a g e of its capacity to remain unaffected by small but deliberate variations in procedural parameters listed in the procedure documentation and provides an indication of its suitability during normal usage [6]." ...
... The ruggedness/robustness of the method was checked afterdeliberately altering the following parameters: composition ofthe mobile phase, mobile phase flow rate, injection volume,column temperature and detector wavelength (Mulholland, 1988). The results showed no significant statistical differences between various altered parameters with respect to those which were received initially i.e. retention time, relativeretention time (RRT), resolution and number of plates ( Table 2). ...
Background: Tapentadol hydrochloride (TAP) is a novel opioid that binds and activates the opioid receptor in the central nervous system to modify the approach our body interprets pain. It has a dual mechanism of action (mu opioid-receptor agonist and noradrenaline reuptake inhibitor), this feature makes it an attractive member of the opioid class. Objective: The aim of the present study was to develop and validate a simple, rapid, selective, sensitive, accurate and precise High-Performance Liquid Chromatography (HPLC) with UV detection method to quantify TAP in rat plasma. Material and methods: Different analytical parameters, such as linearity, accuracy, precision, specificity with intentional degradation, the limit of detection and limit of quantification (LOQ), were determined according to the ICH guidelines. The chromatographic separation of tapentadol hydrochloride was achieved with LC-2010 HT column using a mobile phase, potassium phosphate buffer: acetonitrile (50:50 v/v) at a flow rate of 1.0 ml/min. using a UV detector set at 272 nm with a continuous run up to 5 min. Plasma samples were processed using acetonitrile as a precipitating agent to extract the drug. Results and conclusion: The linearity for tapentadol hydrochloride was found to be 100-1000 ng/ml with regression coefficient (r2)> 0.9970. The recovery ranged from 98.9 to 100.8% for the drug with a relative standard deviation (%RSD) of <2%. Stability analysis revealed that the drugs remained stable for sufficient time. The limit of quantification in plasma for tapentadol hydrochloride was found to be 10ng/ml. The mean recovery was obtained at 98.96%. The chromatographic runs were specific with no interfering peaks at the retention times of the analytes confirmed by the experiments. The method can be used to perform pharmacokinetic and bioequivalence studies in rat blood/serum.
... According to the results obtained, the reduction was very efficient with an average recovery value of 101.8% for 50 nM, 101.1% for 150 nM and 100.3% for 400 nM spiked samples. The robustness of the method was assessed by applying a saturated factorial fractional design of experiments [36], because many factors can be studied with just a few experiments. The experimental factors selected were the same as in the BWD, but also including other parameters that could affect the quality of results, such as the pH of the buffer solution (pH) and the column temperature (T c ). ...
Atmospheric iodine plays a relevant role in climate change. Bearing in mind that most of this iodine comes from the oceans, analytical methods capable of determining iodine in a challenging matrix as seawater are necessary. In this work, the first method capable of direct determination of total inorganic iodine in seawater at subnanomolar level based on mixed-mode liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) without any sample treatment is presented. Analytical characteristics of the developed method were studied in terms of linear range, limits of detection and quantification, precision, trueness, matrix effect, and robustness. The detection limit for iodide was as low as 0.16 nM, injecting 5 μL of seawater without any sample treatment and the working linear range of four orders of magnitude was wide enough to cover the broad concentration range observed in seawater samples. Average values for repeatability and intermediate precision were 4.1% and 8.1%, respectively. The suitability of the method was demonstrated through its application to the analysis of several types of samples, including seawater samples taken at different locations along the Spanish Mediterranean coast and some domestic iodized salts. According to the results obtained, the method developed is rapid, easy to apply and to be automated, avoids sample treatment and requires only few microliters of sample. Furthermore, it has a low detection limit and allows the quantification of inorganic iodine over a wide concentration range.
... For this reason, the authors may follow some review papers, book chapters, books, and guidelines for further reading that they can find in the references. [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] ...
... Measured the capability of the method to stay unaffected by small variations in the parameters [59][60][61][62][63]. From Table 4, it was found that small changes in reagent volume and the type of the instrument had no respectable changes in the results obtained. ...
A simple, fast and accurate spectrophotometric method had been developed to determine lead (II), chromium (III) and barium (II) ions in pure forms and in spiked water samples using thoron (THO) as a reagent forming colored complexes. It was found that the formed complexes absorbed maximally at 539, 540 and 538 nm for Pb(II)-THO, Cr(III)-THO and Ba(II)-THO complexes, respectively. The optimum experimental conditions for these complexes had been studied carefully. Beer's law was obeyed in the range 1–35, 1–70, and 1–45 μg mL− 1 for Pb (II), Cr(III) and Ba(II) ions with THO reagent, respectively. Different parameters such as linearity, selectivity, recovery, limits of quantification and detection, precision, and accuracy were also evaluated in order to validate the proposed method. The results showed that, THO was effective in simultaneous determination of Pb(II), Cr(III) and Ba(III) ions in pure forms and in spiked water samples. Also, the results of the proposed method were compared with that obtained from atomic absorption spectrometry. The isolated solid complexes had been characterized using elemental analysis, X-ray powder diffraction (XRD), IR, mass spectrometry and TD-DFT calculations. Their biological activities were investigated against different types of bacteria and fungi organisms.
... The precision of the dual sensors label response is presented with the reproducibility of the label response, and it has been given as the error bars in Fig. 2 (A and B), where the value of the errors bars were less than 2%, which are excellent for this type of measurements (Miller & Miller, 2010). In addition, the robustness (Miller & Miller, 2010;Mulholland, 1988) of the dual sensors label, the labels were already prepared in a different batch of the label preparation and different days to test the response of the dual sensors. The results show that the sensors give consistent response toward the beef freshness. ...
On-package dual sensors label has been constructed based on two pH indicators, and it has been applied to monitor the beef freshness. The two pH indicators used are methyl red (MR) and bromo cresol purple (BCP). The beef decay could be detected clearly and easily by the dual color indicators, where the MR change from red to yellow, while the BCP change from yellow to purple. The label responses accurately to the beef freshness, in term of its pH change due to the deterioration as shown by both color change at room and chiller temperatures. Similarly with the other parameters, such as the total volatile basic amine (TVBN), total viable count (TVC) and sensory evaluation. These levels were reached at 8 h and 7 days as the threshold of spoilage at room and chiller temperature respectively. Therefore, the label can be used as a simple and practical indicator for freshness monitoring of packaged beef.
... The precision of the sensor response related to the reproducibility of the measurement is shown as the error bars in Figure 2, where error values were smaller than 5%, which is acceptable for this type of measurement [19]. Furthermore, as for the ruggedness or robustness [19,20] of the sticker sensors, they were prepared before they were used in different batches on different days to test the sensor response. Based on our experiment, the sensor showed a consistent response toward beef freshness. ...
A simple sticker sensor has been constructed using litmus paper and tests have been conducted to detect the freshness of beef samples. The results show that the sticker sensor can be used to determine the degree of beef freshness, since the color change of the litmus paper and the quality degradation of the beef during storage time had a similar trend, where the decay of the beef could be detected clearly (when the red litmus paper changed to blue). The sticker sensor reacted accurately to the beef’s freshness in terms of pH change due to beef deterioration from pH 5.61 to 6.24 and from pH 5.67 to 6.02 as shown by its color change in real time at room and chiller temperature respectively. Thus, the sticker sensor can be used as an effective tool for monitoring the microbial quality of packaged fresh meat that correlates with the increased pH of the beef, where the total viable count (TVC) of 5 x 106 cfu/g or 6.698 log cfu/g correlates with a pH of 6.24. These levels were reached at 10 hrs and 7 days at room and chiller temperature respectively. This study provides a foundation for developing a simple sensor for beef freshness.
... However, performing a robustness test late in the validation procedure involves the risk that when a method is found not to be robust, it should be redeveloped and optimized. One can save money and efforts spent atthat pointtherefore the performance of a robustness test has been shifting to earlier points of time in the life of the method [1][2][3][4][5]. According to USP general chapter <1225>, "the robustness of an analytical procedure is a measure 215 | P a g e of its capacity to remain unaffected by small but deliberate variations in procedural parameters listed in the procedure documentation and provides an indication of its suitability during normal usage [6]." ...
... Robustness is generally studied through traditional approaches which suffers major drawbacks like inability to determine complex interactive effects between method variables like pH, column temperature, fl ow rate, buffer concentration, mobile phase composition etc., and is a time consuming process which takes several runs to obtain data as a single variable is changed at each run while other approach being the optimization using quality by design, as per ICH Q8 guidelines, with design of experiments (DOE) leads to ensure success in fi nal method validation. 14, 15 Ye et al. 16 have reported the applicability of DOE and data treatment by JMP ® and Hibbert 17 has reviewed the applicability of DOE in chromatography. In recent years the applicability of DOE and statistical data treatment of years, HPLC data have increased immensely. ...
Objective: To develop and validated a new stability indicating reverse phase high performance liquid chromatography (RP-HPLC) method for analysis of naftopidil (NAF), both as a bulk drug and in formulation. Method: The separation was achieved using a C18 GRACE column (250 mm × 4.6 mm i.d., 5 μm particle size) and gradient mobile phase system consisting of (A) 10 mM of ammonium acetate buffer pH adjusted to 4.0 with glacial acetic acid and (B) acetonitrile. The flow rate was 1.0 mL/min with UV detection at 284 nm. NAF was subjected to stress conditions like hydrolysis (acid, alkali and neutral degradation), oxidation, photolytic and thermal decomposition. The linearity of the proposed method was investigated in the range of 10-150 μg/mL. Application of design of experiments for the robustness study method was carried out, where in five factors was selected: pH of mobile phase, flow rate, strength of the buffer and column temperature. These factors were examined using JMP@ (SAS Institute) software. Result: The analytical method for NAF was developed and validated at the linearity range of 10-150 μg/mL. The LOD and LOQ were 0.6 and 2.04, respectively and accuracy of analysis was 100.5-101.1%. Conclusion: A robuststability-indicating HPLC assay method was developed using DOE, for the quantitation of NAF in its bulk and tablet dosage forms.
... toluene: ethyl acetate: 6 M ammonia solution (5:6:0.5, 4.8:5.8:0.5 and 5.2:6.2:0.5 v/v/v), chamber saturation time (15, 30 and 45 min), plate activation time (1, 2.5 and 5 min) and volume of mobile phase (11.9, 5.95 and 23.8 mL) (Sethi, 1997;Zeaiter et al., 2004). The ruggedness (Mulholland, 1988) of the method was assessed by comparison of intra-day and inter-day results for the assay of ropinirole performed by two analysts in the same laboratory. ...
Stability-indicating high-performance thin-layer chromatographic (HPTLC) method for the analysis of ropinirole HCl was developed and validated as per the ICH guidelines. The method employed the mobile phase and toluene-ethyl acetate-6 M ammonia solution (5:6:0.5, v/v/v) was optimized with the help of a design expert. Densitometric analysis of ropinirole HCl was carried out in the absorbance mode at 250 and 254 nm. Compact spots for ropinirole HCl were found at R f value of 0.58 ± 0.02. The linear regression analysis data for the calibration plots showed R (2) = 0.9989 ± 0.0053 with a concentration range of 100-3000 ng spot(-1). The method was validated for precision, accuracy, ruggedness, robustness, specificity, recovery, limit of detection (LOD) and limit of quantitation (LOQ). The LOD and LOQ were 12.95 and 39.25 ng spot(-1) respectively. Drug was subjected to acidic, alkaline, oxidative, dry heat, wet heat and photo degradation stress. All the peaks of degradation products were well resolved from the standard drug peak with significant difference of R f. The acidic and alkaline stress degradation kinetics of ropinirole, were found to be in first order, showing high stability (t 1/2, 146.37 h(-1); t 0.9, 39.11 h(-1)) in the acidic medium and low stability (t 1/2, 97.67 h(-1); t 0.9, 14.87 h(-1)) in the alkaline environment.
... When screening at only the extreme levels (−1, +1), the intermediate optimum is ignored. A possibility to screen the factor(s) at three levels is by using so-called reflected designs (4,32,(63)(64)(65). Reflected designs are duplicated two-level full factorial, FF, or PB designs. ...
In the lifetime of a chromatographic method different stages can be considered. Once a technique has been selected, a method is developed (method development) and optimized (method optimization). An optimized method is a method that shows a sufficient resolution of the relevant peaks that gives acceptable and preferably robust results in a short analysis time. Before starting with method validation often a robustness or ruggedness test is performed. In a robustness/ruggedness test, the influence of small variations is evaluated in the procedure on the performance of the method. These small variations are deliberately introduced and represent variations that could occur when a method is transferred—for example, from one laboratory to another. While in the optimization step the studied responses are related to the quality of the separation, in a robustness test the quantitative aspects of the method are reviewed, and responses related to the separation are only examined in a second instance.
... The first one was proposed by Youden (51), in which a Plackett-Burman design and the appropriate statistical treatment was used. The following proposed methodologies, which are based on the Youden procedure, use different experimental designs (52)(53)(54) or different statistical interpretations (55,56). A review covering the use of robustness and ruggedness in Analytical Chemistry has been edited (57) and a guidance for Robustness/Ruggedness Tests in Method Validation has been published (58). ...
The knowledge of the robustness/ruggedness of analytical processes is an essential feature in the analytical validation. The definitions and the different aims of such studies are given. A dual general procedure for checking robustness/ruggedness in both intrinsic and extrinsic validation is presented and the different steps are discussed. The denomination of “inertia study” is introduced to designate this methodology in extrinsic validation and precision and trueness tests are proposed inside the inertia study. Several experimental designs, depending on the goal of robustness/ruggedness study and the type of variables (quantitative or qualitative) are considered.
... If the response surface displays saddle points or other regions with sudden changes, it is generally tried to avoid such regions. Consequently, one can assume sparsity of factor effects, which means that the interaction effects are usually considered negligible, as has also been shown in practice [29]. ...
Robustness tests are usually based on an experimental design approach. As designed experiments generally lead to a large variability among the results, erroneous results are often not readily detected. As a consequence, the ordinary least squares (OLS) estimates of the effects of the robustness test can be biased. Here, two robustness tests are studied, which both contain a suspicious result. Moreover, simulated datasets are considered to examine the influence of the extent of the outlier as well as the influence of multiple outliers. On the one hand, different methods are applied to inspect the results of the experiments for outliers: the half-normal plot of the OLS residuals, the normal probability plot of the effects and a method, which is based on experimental design reconstruction. On the other hand, two robust regression methods are applied to calculate the effects with a minimum influence of possible outliers. The different methods are compared and it is evaluated under which circumstances they can be applied.
... A possibility to screen the factor(s) at three levels is by using so-called reflected designs [4,32,[63][64][65]. Reflected designs are duplicated two-level full factorial, fractional factorial, or Plackett-Burman designs. ...
... Robustness can be described as the ability to reproduce the (analytical) method in different laboratories or under different circumstances without the occurrence of unexpected differences in the obtained result(s), and a robustness test as an experimental set-up to evaluate the robustness of a method. The term ruggedness is frequently used as a synonym [2][3][4][5]. Several definitions for robustness or ruggedness exist which are, however, all closely related [1,6 -10]. ...
This chapter presents the use of robustness and ruggedness in analytical chemistry. A ruggedness test is a part of method validation and can be considered as a part of the precision evaluation. Ruggedness is related to repeatability and reproducibility. Certain interpretation methods to identify the significant factors in a ruggedness test use criteria based on results for repeatability or reproducibility. The validation of analytical methods is becoming increasingly important, particularly in the pharmaceutical industry. This is due among others to the regulations imposed by the drug regulatory agencies. The ruggedness testing should be performed for nearly all analytical methods used in pharmaceutical and biopharmaceutical analysis. Two types of precision are usually distinguished—namely the repeatability and the reproducibility. The result of a ruggedness test indicates how tightly controlled the experimental factors should be. The detection of factors that heavily influence the results of a method leads eventually to a more reproducible method.
The current studies endeavour to develop and validate a simple, sensitive and robust reversed- phase (RP) high-performance liquid chromatography (HPLC) methodology for quantitative estimation of voriconazole (VRC), following systematic principles of Analytical Quality-by-Design (AQbD). A comprehensive risk assessment plan, followed by factor screening studies employing Taguchi design, was executed to select “vital few” critical method parameters, influencing critical analytical attributes. Subsequently, response surface optimisation studies were conducted on the identified critical method parameters, viz., mobile phase ratio and flow rate, with chosen critical analytical attributes, viz., peak area, theoretical plate count, retention time and peak tailing, employing a Central Composite Design. The design space, demarcating the optimal method operating conditions, was identified using graphical optimisation and subsequently validated employing Monte Carlo simulations. The optimal solution consisted of acetonitrile and acetic acid solution (0.05 %, pH 4) as mobile phase (50:50 v/v), flow rate of 1 mL/ min with a detection wavelength of 256 nm, exhibiting linearity between 0.1 and 50 μg/mL in methanol and Hanks balanced salt solution. Validation studies confirmed precision (97.78-103.42 %), accuracy (98.99-102.34 %), and suitability of the developed analytical method for effective quantification of VRC with high sensitivity (LOD: 0.031 μg/mL; LOQ: 0.093 μg/mL). The validated analytical technique was successfully ratified for quantification of VRC in drug nanoformulations too. In a nutshell, the application of AQbD and Monte Carlo simulations aided in developing a robust analytical method for the estimation of VRC per se as well as in drug nanoformulations.
There are a great many experimental parameters that can be varied to influence the separation selectivity and performance achieved for a CE method. Conventionally when attempting to assess the impact of each of these parameters each factor may be varied sequentially. This “uni-variate” approach typically involves holding all parameters constant and varying one parameter at a time and measuring method responses such as resolution and analysis times. For example, a sequence of injections may be performed to assess the impact of pH, followed by a further injection series to assess impact of ionic strength and further sequences to assess the influence of factors such as buffer additives, organic solvent content, temperature, sample concentration etc. This step-by-step approach, although widely used, involves a large number of independent analyses, and could be replaced by statistically designed experimental protocols in which several factors are simultaneously varied. These multi-variate experimental design approaches have advantages in terms of reductions in the number of experiments, improved statistical interpretation possibilities, and reduced overall analysis time requirements.
Analytical chemistry, specifically electroanalytical chemistry, plays an important role in many branches of chemistry, biochemistry, pharmacy, food productions, life science, environment, etc. Many decisions are based on the results of quantitative analyses, and it is important to be aware of the quality of the results whenever analytical or electroanalytical methods are used. Analytical properties are quality indicators for a variety of systems, objects, tools, and outputs involved in chemical or biochemical measurements that allow one to compare and validate both analytical processes and the results that they provide. The objective of any analytical measurements is to obtain consistent, reliable, and accurate data. Validated analytical methods play a major role in achieving this goal. Analytical method validation is a major issue in the pharmaceutical industry for controlling drug quality, development, and registration. Simply, it is used to justify the analytical method used, in other words, to show that the method accomplishes what is claimed or intended. Validation is required for the development of new analytical methods, methods submitted as a part of a new drug application, bioequivalence and bioavailability studies, and for the analysis of drugs as raw material or in their dosage forms. Likewise, all laboratory tests must be validated before being introduced for patient testing to insure that the values reported will meet clinical expectations with a desired degree of reliability. Revalidation should be required, to a less or greater degree, following any change in reagents, supporting electrolytes, instruments, or experimental conditions. Evaluation and validation of an electroanalytical method performance is required to assess the degree of error expected due to inaccuracy and imprecision and to confirm that the degree of error meets the anticipated laboratory or clinical requirements. The procedures recommended for method validation differ with the type of test and the anticipated use. Experiments must be designed so that the correct data are obtained. The appropriate statistical methodologies should be used to correctly estimate errors with sufficient precision and to make the right decision about method’s validity.
This paper describes validation of an OPLC procedure developed for the determination of aflatoxins B1, B2, G1, and G2 in wheat. Samples were extracted with 9:1 (v/v) acetonitrile-water and the extracts were filtered and evaporated to dryness. OPLC was performed with chloroform-toluene-tetrahydrofuran, 15 + 15 + 1 (v/v), as mobile phase. Before the separation an OPLC prewashing step was necessary; this eliminated the time-consuming and costly clean-up steps of other chromatographic methods. The validation procedure included tests on specificity and determination of the retention factor (RF), resolution (RS), asymmetry (AS), linearity, accuracy, precision, detection limit (DL), and quantitation limit (QL) of the method. Depending on the aflatoxin analyzed the results of the investigation were: 0.20 < RF < 0.60, RS > 1.7, 0.85 < AS <1.1, recovery > 84%, RSD < 10%. The DL of the method was 0.018, 0.100, 0.15, and 0.14 ng for aflatoxins G2, G1, B2, and B1, respectively. A calibration curve was constructed for each aflatoxin by plotting spot area against amount of aflatoxin applied to the plate. The robustness of the procedure was also determined and found be critically dependent on the type of plate used (TLC or HPTLC).
The optimal separation of antiarrhythmic new drug chlorobenzyltetrahydroberberine (CBTHB) and its related substances berberine hydrochloride (B) and tetrahydroberberine ( THB ) by capillary electrophoresis was reported . The effect of main operating parameters on the migration behaviour of analytes was discussed systematically. In a coated silica capillary (20 cm × 25 μm), the berberine hydrochloride, tetrahydroberberine, chlorobenzyltetrahydroberberine and internal standard quinine dihydrochloride could be separated within 6.0 min in the following optimum conditions: 80 mmol/L phosphate buffer at pH 2.5 containing 20% ethanol, 10 kV applied voltage, electromigration injection 4 kV × 6 s, 230 nm on-line detection and 25°C capillary temperature. The ruggedness test was evaluated based on variations of buffer pH value, ethanol content and capillary temperature, and the factors of ethanol content and capillary temperature would affect significantly electrophoretic separation.
This chapter discusses quality assurance in environmental analysis. Environmental management depends on information on a wide range of compounds. Unreliable or inaccurate information may lead to large economic losses, such as sanitation measures that may be taken needlessly, or to unacceptable risks for the environment or human health if necessary measures are not taken. It is essential that the quality of information on the environment is adequate. Quality assurance of environmental analyses represents an aspect in the quality management of the overall process that provides information. The chapter provides a brief analysis of the information requirement. The assessment of this information requirement is the start of a chain of activities that generates the information pursued. Environmental management increasingly takes measures to prevent problems and laboratory tests are necessary in this context. Thus, the chapter also deals with quality management in the laboratory.
A stability-indicating reversed-phase high performance liquid chromatographic (RP-HPLC) method has been developed and validated for simultaneous estimation of atorvastatin calcium and ezetimibe for their multicomponent dosage form. The proposed RP-HPLC method utilizes a 125 mm x 4.6 mm i.d 5 μm Phenomenex C-18 column at ambient temperature; the optimum mobile phase consists of acetonitrile and 0.4% v/v triethylamine (pH adjusted to 5.5 with ortho-phosphoric acid) in the ratio of 55:45, v/v respectively, flow rate of 1.0 ml/min. Measurements were made at a wavelength of 231 nm. Multicomponent dosage form was exposed to thermal, photolytic, hydrolytic and oxidative stress. No co eluting, interfering peaks from excipients, impurities were observed for the degradation products and hence the method was found to be specific. The method was linear in the range of 5-25 μg/ml for atorvastatin calcium and ezetimibe. The mean recoveries were 98.82% and 98.72% for atorvastatin calcium and ezetimibe respectively. The method was validated for linearity, range, precision, accuracy, specificity, selectivity, intermediate precision, ruggedness, robustness, solution stability and suitability.
A stability-indicating high-performance liquid chromatography (HPLC) procedure was developed for the determination of metformin HCl (MTH), atorvastatin calcium (AC) and glimepiride (GP) in combination and their main degradation products. The separation and quantization were achieved on a 5-µm Qualisil gold, C18 column (4.6 mm × 250 mm). The mobile phase selected was phosphate buffer (pH 2.9)-organic phase in proportion of 70:30. Organic phase consisted of methanol-acetonitrile (90:10) at a flow rate of 1 mL/min and detection of analytes was carried out at 230 nm. The method exhibited good linearity over the range of 10-60 µg/mL for MTH, 2-20 µg/mL for AC and 5-30 µg/mL for GP. Square of the correlation coefficients was found to be >0.999. Various stress degradation studies were carried out in combination as per International Conference of Harmonization (ICH) guidelines for 4 h. The recovery and precision were determined in terms of intraday and interday precisions and expressed as relative standard deviations. These were <1 and <2%, respectively. Finally, the applicability of the method was evaluated in nanoparticle analysis of MTH, AC and GP as well as in stability studies of nanoformulation.
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An accurate, sensitive, precise, rapid, and isocratic reversed phase HPLC (RP-HPLC) method for analysis of ropinirole in the bulk drug and in pharmaceutical preparations has been developed and validated. The best separation was achieved on a 250 mm × 4.6 mm i.d, 5-μm particle, C 18 reversed-phase column with methanol-0.05 m ammonium acetate buffer (pH 7) 80:20 (v/v) as mobile phase, at a flow rate of 1 mL min-1. UV detection was performed at 250 nm. The method was linear over the concentration range 0.2-100 μg mL-1 (r = 0.9998), with limits of detection and quantitation of 0.061 and 0.184 μg mL-1, respectively. The drug was subjected to oxidation, hydrolysis, photolysis, and heat as stress conditions. Degradation products resulting from the stress did not interfere with detection and assay of ropinirole and thus the method can be regarded as stability-indicating. The method can be used for quality-control assay of ropinirole.
A simple, precise and stability-indicating thin-layer chromatographic method for estimation of ropinirole HCl was developed and validated as per ICH guidelines. The mobile phase consisted of acetone–cyclohexane–6 M ammonia solution (8:5.5:0.5, v/v/v). Scanning the drug was done at 250 nm. Compact spots for ropinirole HCl were found at an R
f value of 0.51 ± 0.002. The linear regression analysis data for the calibration plots showed good linear relationship with R
2 0.9976 ± 0.0011 in the working concentration range of 100–3,000 ng spot−1. The method was validated for precision, accuracy, ruggedness, robustness, specificity, recovery, limit of detection (LOD), and limit of quantitation (LOQ). The LOD and LOQ were 12.89 and 42.53 ng spot−1, respectively. The drug was subjected to degradation; the peaks of degradation products were well resolved from the standard with significantly different R
f values. Hence, this method can be used for quality control assay of ropinirole.
Process parameters for the extraction of glycolipids by scCO2 from Lactobacillus plantarum B-01 were optimized by carrying out a two-level fractional design experiment. The highest glycolipids extract yield was obtained at 250bar, 40°C, CO2 flow rate, 10g/min and hydrometanolic co-solvent, 9.3% (v/v). In the lipid extracts the carbohydrate moiety of glycolipids and in phospholipids phosphorus moiety were quantified and compared to the reference material isolated by classical organic solvent extraction. By TLC four groups of glycolipids were identified. A two step scCO2 extraction process was established, whereby the second step yielded seven times more of glycolipids and four times more of phospholipids than in the extracts obtained with organic solvents. Prolonged scCO2 extraction to 9h resulted in 620μg of glycolipids and 875μg of phospholipids in a mg of extract in comparison to (1.5h) 55μg and 15μg of glycolipids and phospholipids, respectively.
Accelerated solvent extraction (ASE) was developed for the rapid extraction of cocaine and benzoylecgonine from coca leaves. Several parameters such as the nature of the extracting solvent, the pressure, temperature, extraction time, addition of alkaline substances, and sample granulometry were investigated to find the best extraction conditions. A central composite design was used to optimise critical parameters (pressure, temperature, and extraction time) and to assess the robustness of the extraction method. It was demonstrated that the extraction method was perfectly robust around optimal conditions (20 MPa, 80°C, and 10 min). Determination of both cocaine and benzoylecgonine in coca leaves was carried out by means of gas chromatography with flame ionisation detection (GC-FID) or capillary electrophoresis with UV detection (CE-UV). With the latter, separation of both compounds was achieved in less than 4 min with the use of a short-end injection procedure.
For a specific test method, ruggedness experiments are used to determine experimental conditions that significantly influence the measurement results and to determine how closely these conditions must be controlled to reduce their influence. In this paper, the results of ruggedness experiments using hot box test methods (ASTM C 1199, C 1363, and ISO 12567-1) that are used for the thermal transmittance measurement of fenestration systems are presented. A general procedure of ruggedness evaluations was conducted on the University of Massachusetts Research Calibrated Hot Box. Using ASTM E 1169, a Plackett-Burman (P-B) design with eight experiments was used to investigate the effects of the seven most important experimental conditions. Two experimental conditions, the weather side and metering side fan speeds, were found to have the most significant effects on fenestration systems thermal transmittance measurement results.
The search for sources from which small changes have an influence on the response of an analytical system, the ruggedness test, has become an essential part of a validation study. However, the `definition' of ruggedness and how to perform a ruggedness test are still ambiguous. In this paper a strategy is proposed to perform a ruggedness test for chromatographic methods. Because the strategy is based on saturated designs, ANOVA cannot be applied to estimate the error variance. Therefore a graphical technique, the half-normal plot, is used to detect influential effects. In order to identify influential effects, the algorithms of Lenth and Dong are applied to four different examples. If a main effect is found to be significant, the confounded two-factor interactions are resolved by a follow-up design. The strategy of this sequential design is implemented in a SAS® software application called TRIC, testing ruggedness in chromatography.
A method is presented for the determination of Al, Si, Fe, Ti, V, Mo, Ni and Co in catalysts used in different processes in the oil and petrochemical industries. The method is based on the dissolution of samples in a nitric and hydrofluoric acid mixture aided by microwave energy. After dissolution of the samples, the elements of interest are present in a matrix of fluoboric acid (60% v/v), where they are determined by flame atomic absorption spectrometry (FAAS). Interference studies for the FAAS determination of elements based on experimental statistical factorial designs are also presented. Recoveries better than 96% for all elements were obtained with relative standard deviations lower than 5% for major components and lower than 6% for minor components.
Using Response Surface Methodology (RSM) and based on sequential experimental Doehlert designs, a Solid-Phase Spectrophotometric determination of cadmium, at μg/l level, with 4-(2-pyridylazo)resorcinol in the presence of potassium iodide, has been established. The method has been characterized and validated by obtaining the performance characteristics. The IUPAC detection limit is 1.5μg/l and reproducibility (RSD) in the determination of 76.0μg/l is 3.4%. Robustness was established using two-level saturated fractional factorial designs. Selectivity was studied and trueness was confirmed with house reference materials.
An HPLC method is defined as a specific setting of the physical, chemical and data processing parameters which control the chromatographic analysis. During the development or validation of a method no special attention is usually given to the data processing parameters. However, it turns out that HPLC methods for complex samples can be very sensitive to minor changes in certain numerical integration parameters such as the threshold. A series of statistically designed Chromatographic runs for a dyestuff with a very large number of peaks is presented where the interpretation of the data depends crucially on which threshold parameter value is chosen. It is therefore recommended that for development or validation of an HPLC method, especially in the context of quality control of complex substances, greater attention should be paid to the integration parameters.
An accurate, sensitive, precise, rapid and isocratic reversed-phase HPLC (RPHPLC) method for analysis of buspirone in the bulk drug and in solid dosage formulations has been developed and validated. The best separation was achieved on a 250 mm × 4.6 mm i.d., 5-μm particle, RP C 18</ sub> column with 70:30 ( υ/υ ) methanol-0.01 m </ span> sodium dihydrogen phosphate buffer (pH 3.5) as mobile phase at a flow rate of 0.8 mL min -1 . UV detection was at 244 nm. Response was a linear function of concentration over the range 0.05-20 μg mL -1 ( r = 0.9998) and the limits of detection and quantitation were 3.7 and 11.3 ng mL -1 , respectively. The method was validated in accordance with ICH guidelines. The drug was subjected to oxidative, hydrolytic, photolytic, and thermal stress. Degradation products produced as a result of this stress did not interfere with detection of buspirone and the assay can thus be regarded as stability-indicating. The method was used for quantification of buspirone in commercial buspirone tablets and to check content uniformity. The excipients present in the formulation did not interfere with the assay. The method is suitable for application in quality-control laboratories, because it is simple and rapid with good accuracy and precision.
Application of the ICH procedure to the validation of a chiral capillary electrophoresis method for the quantification of the major stereoisomeric impurity of galantamine hydrobromide drug substance is discussed. Because the reproducibility of capillary electrophoresis as an analytical technique is often questioned, special emphasis was put on the robustness evaluation. The method was found to be accurate, precise and adequately robust, and consequently able to be transferred with confidence to other quality-control laboratories for the successful performance of the procedure.
A stability-indicating reversed-phase high-performance liquid chromatography (RP-HPLC) method has been developed which can separate and accurately quantitate Mebeverine hydrochloride (MEB) and Chlordiazepoxide (CPZ) in commercial tablets. The method has shown adequate separation for MEB and CPZ from their degradation products and main impurities of CPZ whether in pure forms or in commercial tablets. A gradient mobile phase system consisting of (A) water and (B) methanol was used with Phenomenex&® Luna C18 analytical column (250mm x 4.6mm i.d., 5μm ps). Quantitation was achieved with UV detection at 254 nm, based on peak area. MEB and CPZ were subjected to acidic, basic hydrolysis and oxidative degradation to apply stress conditions. The linearity of the proposed method was investigated in the range of 40-130 μg ml-1 (r = 0.9987) for MEB and 8-22 μg ml-1 (r = 0.9991) for CPZ. The limits of detection were 4.77μg ml-1 and 0.71μg ml-1 for MEB and CPZ, respectively. The limits of quantitation were 14.44 μg ml-1 and 2.14 μg ml-1 for MEB and CPZ, respectively. Degradation products of MEB, CPZ and impurities of CPZ did not interfere with the detection of MEB and CPZ. The proposed method can thus be considered as a stability indicating assay.
The fluorescence emission from the lithium/1,4-dihydroxyanthraquinone system shows a great enhancement in the presence of certain water-miscible solvents. This is justified from the donicity (nucleophilic properties) of the solvent that facilitates the solvation of the lithium cation in solution and the stabilization of an nondissociated ion-pair between the solvated lithium cation and the 1,4-dihydroxyanthraquinonate anion. A very sensitive analytical method was proposed for the spectrofluorimetric determination of lithium based on its reaction with 1,4-dihydroxyanthraquinone (quinizarin) in a dimethylsulfoxide medium (90%) and in presence of sodium hydroxide. The fluorescence is measured at an excitation wavelength of 602 nm and an emission wavelength of 670 nm and it is stable at 25°C at least 6 h. The calibration curve is linear over the concentration ranges of 2–40 μg/l of lithium in an aqueous matrix and 3–50 μg/l in a serum matrix; the RSD's in the determination of 20 μg/l of Li were 2.6% and 3.2%, respectively. The proposed procedure was satisfactorily applied to the determination of lithium in drugs, dietetic products and human serum.
This study presents an example of method validation, applied to an IIR-RP-HPLC method, developed for the separation of nine PPPs (Priority Pollutant Phenols). The parameters of both primary validation (accuracy, precision, response linearity, detection. and quantitation limits,) and of secondary validation (method ruggedness) are evaluated. The glossary used in literature for ruggedness and robustness is discussed. The robustness test is carried out with respect to five chromatographic conditions, both depending on analyst accuracy (concentration of the ion-interaction reagent, concentration of the organic modifier, and pH of the mobile phase) and on instrumentation (column temperature and flow-rate).The effect of the experimental factors is studied by a fractional factorial design and mathematical models are built, that correlate the chromatographic retention to the experimental factors and to their interactions. The cross-validated models can usefully be employed to evaluate for each studied variable the region of acceptable ruggedness for any given confidence level.
A twelve run screening factorial experimental design was used to study the instrumental robustness of an HPLC weight percent assay for a fermentation derived pneumocandin B0. The factors varied were the instrumental settings of wavelength, injection volume, flow rate, mobile phase composition, column temperature, and column lot. The measured responses were the fundamental liquid chromatographic parameters of: retention time (RT); capacity factor (k′); theoretical plates (N); tailing factor (T); and resolution (Rs). The effect of each factor on the responses was calculated and significance determined by analysis of variance (ANOVA).
High-performance liquid chromatography (HPLC) and chemometric methods were applied to the simultaneous determination of the
two nonsteroidal antifungal drugs, miconazole (MIC) and nystatin (NYS). The applied chemometric techniques are multivariate
methods including classical least squares, principal component regression and partial least squares methods. The ultraviolet
(UV) absorption spectra of the standard solutions of the training and validation sets in methanol are recorded in the range
of 280–320 nm at 0.2-nm intervals. The HPLC method depends on reversed-phase separation using a C18 column. The mobile phase
consists of a mixture of methanol–acetonitrile–ammonium acetate buffer (pH 6; 50 mM) (60:30:10 v/v/v). The UV detector was
set at 230 nm. The developed methods were validated and successfully applied to the simultaneous determination of MIC and
NYS in their tablets. The assay results obtained using the chemometric methods were statistically compared to those of the
HPLC method and good agreement was observed.
A very sensitive analytical method is proposed for the determination of lithium based on the reaction of Li+ ion with 1,4-dihydroxyanthraquinone (Quinizarin). In dimethylsulfoxide medium (90%) and in the presence of sodium hydroxide and sodium carbonate, a bluish-violet color (λmax = 601 nm) develops and is stable over a period of 30 min to 2.5 h. The NaOH and Quinizarin concentrations were optimized simultaneously using the response surface methodology from sequential experimental Doehlert designs. Beer's law is obeyed in the concentration range 14–250 ppb Li+ in aqueous and serum matrices, and the errors (RSD) in the determination of 100 ppb Li+ are 4.0% and 3.9% respectively. The proposed procedure was satisfactorily applied to the determination of lithium in drugs and human serum (no deproteinization is required).
The planning of collaborative trials of analytical methods is considered, and the use of established statistical procedures for evaluating data generated by such trials is discussed.
When analytical methods are developed for use in many different laboratories it is particularly important to be aware of the sensitivity of the method to variations in the specified conditions. It was the aim of this research to establish a ruggedness test which would enable the used: (1) to decide which variables (flow-rate, etc.) have a significant effect on the chromatographic results; (2) to define objective system suitablity parameters. The test uses a system of fractional factorial experiments, based on the Plackett-Burman design schemes. The test calculates main effects and standard errors. It has also been expanded to include the methodology to test two extreme values for each variant.Both eight-experiment and sixteen-experiment Plackett-Burman schemes were investigated initially, using the well-understood high-performance liquid chromatographic graphic analysis of aspirin and salicylic acid. Several variables were tested, including flow-rate, acid type, mobile phase composition, detectio wavelength, detector atenuation and response time. Both qualitative (retention time, plate count, peak symmetry, etc.) and quantitative (peak area, height, concentration, etc.) parameters were checked for variations.By testing the ruggedness of chromatographic methods in this manner, the analyst can achieve a comprehensive understanding of the limitations and stability of the methodology employed.
Method validation guidelines, which provide an organizational structure for the design and evaluation of a validation procedure, are presented for a wide range of pharmaceutical applications. The validation guidelines are based on the analyte concentration/sample matrix combination to which the method will be applied. These guidelines include the selection of appropriate validation parameters, design considerations for evaluation, and a discussion of acceptance guidelines for the determination of acceptable method performance. A set of tables is included which illustrates the selection and testing procedure and tailors the entire validation process to the specific characteristics of the determination to be made.
Statistical Manual of the AOAC
- W J Youden
- E M Stiener
W. J. Youden and E. M. Stiener, Statistical Manual of the AOAC, AOAC, Arlington, VA, 1975, p. 33. G. Wernimont, ASTM Standardisation News, March (1977) 13-16.