HPLC methods development and validation

A typical example of HPLC method development and validation was provided by Boneschans et al. [9]. They developed an HPLC method for piroxicam benzoate and its major hydrolytic degradation products, piroxicam and benzoic acid. The authors utilised a robust stationary phase (Phenomenex Luna, Cig), with an optimised mobile phase comprising of acetonitrile/water/acetic acid (45/7/8 v/v), and a flow rate of 1.5 ml/min. The operating pH of the mobile phase (pH 2.45) was selected on the basis that it is ca. 2 pH units from the pKa of the drug, and hence reasonably insensitive to changes in mobile-phase preparation. The injection volume was 20 pi with a detection wavelength of 254 nm. They utihsed... 

M. Mulholland, N. Walker, J.A. van Leeuwen, L. Buydens, H. Hindriks and P.J. Schoenmakers, Expert systems for method development and validation in HPLC. Microchim. Acta, 2 (1991) 493-503. 

The primary object of this book is to provide the HPLC practitioner with a handy guide to the use of HPLC for analyzing pharmaceutical compounds of interest. This means familiarizing the practitioner with the theory, instrumentation, regulations, and numerous applications of HPLC. This handbook provides practical guidelines using case studies on sample preparation, column or instrument selection, and summaries of best practices in method development and validation, as well as tricks... 

Unfortunately, few analytical methods are developed in a perfect world. A more likely situation has method development and validation scheduled on a tight time line. Using an HPLC assay and related compounds method for a novel DS as an example,Table 5 outlines a realistic development scenario that would provide for a method that could be successfully validated and utilized. 

The author thanks the following scientists at DuPont Crop Protection and Battelle, Geneva Research Centres, for developing and validating some of the methods summarized in this article C.R. Powley for the soil method by HPLC/MS/MS J. J. Stry, SJ. Hill and PR Maliszewski for the water method by HPLC/MS/MS and K.M. Jernberg, B. Francon, M. Jetzer, C. Steiner, L. Dubey and H. Mattou for the independent laboratory validations of the Multiresidue Method 2 for crop samples. 

The final step of method development is validation of the HPLC method. Optimisation of chromatographic selectivity [110], performance verification testing of HPLC equipment [591], validation of computerised LC systems [592] and validation of analysis results using HPLC-PDA [34] were reported. The feasibility of automated validation of HPLC methods has been demonstrated [593]. Interlaboratory transfer of HPLC methods has been described [594]. 

The near-IR technique has been used very successfully for moisture determination, whole tablet assay, and blending validation [23]. These methods are typically easy to develop and validate, and far easier to run than more traditional assay methods. Using the overtone and combination bands of water, it was possible to develop near-IR methods whose accuracy was equivalent to that obtained using Karl-Fischer titration. The distinction among tablets of differing potencies could be performed very easily and, unlike HPLC methods, did not require destruction of the analyte materials to obtain a result. 

Fogli et al. developed and validated an HPLC method with fluorescence detection for simultaneous routine TDM of anthracyclines and their metabolites.27 They coupled a Waters LC Module I Plus system equipped with a WISP 416 autosampler with a Model 474 scanning fluorescence spectrophotometer. The stationary phase was a Supelcosil LC-CN column (250 x 4.6 mm, 5 /um particle size) with a /iBondapak-CN guard column. The mobile phase consisted of 50mM monobasic sodium phosphate buffer and acetonitrile (65 35 v/v), adjusted to pH 4.0 with phosphoric acid. The flow rate was 1 mL/min. The fluorescence detection was set at excitation wavelengths of 233, 254, and 480 nm and at an emission wavelength of 560 nm. 

Sirolimus is a potent immunosuppressive agent. To prevent thrombocytopenia and hypercholesterolemia, optimize efficacy, and reduce organ rejection, assays were developed to monitor concentrations of sirolimus in the whole blood of patients under treatment.40"12 Wallemacq et al.43 developed and validated a simple high-throughput HPLC-MS/MS method to routinely monitor sirolimus... 

From Analytical Chemists, a partial answer to this problem was the development and validation of new methods that permit an improvement in terms of productivity ( high-throughput ), sensitivity and selectivity, especially using very recent hyphenated analytical assays, such as HPLC-MS/MS, GC-MS/MS or further complex couplings, that can provide more complete information in a single analysis. 

Roda A. Sabatini L. Barbieri A. Guardigli M. Locatelli M. Violante F.S. Rovati L.C. Persiani S. Development and validation of a sensitive HPLC-ESI-MS/MS method for the direct determination of glucosamine in human plasma. Journal of Chromatography B, 2006, 844, 119-126. 

Tim Wehr is Staff Scientist at Bio-Rad Laboratories in Hercules, California. He has more than 20 years of experience in biomolecule separations, including development of HPLC and capillary electrophoresis methods and instrumentation for separation of proteins, peptides, amino acids, and nucleic acids. He has also worked on development and validation of LC-MS methods for small molecules and biopharmaceuticals. He holds a B.S. degree from Whitman College, Walla Walla, Washington, and earned his Ph.D. from Oregon State University in Corvallis. 

Because of antibody-based selectivity, ELISAs are capable of handling samples that are impure or only semipurihed. It is possible to perform ELISAs in a variety of matrices. This is in contrast to other methods such as HPLC that require relatively pure material. During the development and validation of the ELISA method, it needs to be demonstrated that the ELISA is not affected by interfering substances that could be in the test sample, such as buffers, salts, contaminating proteins, and excipients. It also needs to be demonstrated that the conjugated antibody does not bind nonspecihcally to the coated solid phase. 

Traditionally, HPLC, GC-MS, or LC-MS methods were used to monitor the clearance of small-molecule impurities. These analytical techniques often require unique solvents, columns, methods, reagents, detectors, and buffers for each analyte to be quantified. The NMR method, albeit not the most sensitive technique, normally does not have these problems. In this chapter, some examples will be used to demonstrate that NMR is a fast, generic, and reliable analytical technique for solving analytical problems encountered in the development of biopharmaceutical products. The NMR techniques described here require minimal sample handling and use simple standard NMR methods. They can easily be implemented and used for process development and validation purposes. 

A PDA detector provides UV spectra of eluting peaks in addition to monitoring the absorbance of the HPLC eluent like the UVA is absorbance detector. It is the preferred detector for testing impurities and for method development. PDA facilitates peak identification during methods development and peak purity evaluation during method validation. Detector sensitivity was an issue in earlier models but has improved significantly (more than ten-fold) in recent years. ... 

Validation is the process of proving that a method is acceptable for its intended purpose. It is important to note that it is the method not the results that is validated. The most important aspect of any analytical method is the quality of the data it ultimately produces. The development and validation of a new analytical method may therefore be an iterative process. Results of validation studies may indicate that a change in the procedure is necessary, which may then require revalidation. Before a method is routinely used, it must be validated. There are a number of criteria for validating an analytical method, as different performance characteristics will require different validation criteria. Therefore, it is necessary to understand what the general definitions and schemes mean in the case of the validation of CE methods (Table 1). Validation in CE has been reviewed in references 1 and 2. The validation of calibrations for analytical separation techniques in general has been outlined in reference 3. The approach to the validation of CE method is similar to that employed for HPLC methods. Individual differences will be discussed under each validation characteristic. 

A fully automated on-line SPE-HPLC-MS-MS method was developed and validated for the direct analysis of 14 antidepressants and their metabolites in plasma by de Castro et al. [75]. After direct injection of 50pL of plasma without prior sample pre-treatment, gradient RP separation was completed in 14 min, with a sample throughput of 3h. LOQs were estimated to be at 10 ng/ mL. Analytes proved to be stable during sample process with the exception of clomipramine and norclomipramine. 

An HPLC method was developed and validated for the determination and quantitation of both DFC cysteine and glutathione conjugates. This method was based on a cleavage of the disulphide and/or thioester bonds between the metabolites and their conjugate sulphur-containing moiety using dithioerythritol to yield DFC, which was then stabilized by derivatizing to DFC acetamide (91). 

The determination of OXO in Japanese oyster was realized using reversed-phase HPLC. Samples were extracted with LLE and SPE recoveries were 88.3% (193). Oyster samples were homogenized with a phosphate buffer adjusted to pH 7. After centrifugation, supernatants were concentrated using an SPE C-18 cartridge. Before use, the cartridge was activated with MeOH and phosphate buffer. After the sample had been passed, the cartridge was flushed with water and the analytes were eluted with MeOH-orthophosphoric acid (9 1). The eluate was evaporated, and the residues were dissolved in the mobile phase. The method developed was validated and the study of OXO stability was performed. The limits of detection and determination were 10 and 40 ng/ml, respectively. 

MacArthur, R. et al. (2002) Development and validation of an HPLC method for simultaneous determination of intense sweeteners in food stuffs (A01012), DEFRA Central Science Laboratory, York. 

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