High-performance liquid chromatography method validation

In the last twenty years, many of the developed and validated high performance liquid chromatography methods with conventional diode array or fluorescence detectors (DAD, FLD) were improved and substituted by new hyphenation with mass spectrometric instrumentation and/or NMR, especially for the analyses of raw materials derived from Natural sources. The main goal of this coupling is achieved by improvement of selectivity and sensitivity of new instrumental configurations [7], Furthermore, with these configurations it is possible to obtain, in only one analysis, the complete chemical structure elucidation, identification and quantification of targeted compounds. 

This chapter focuses on approaches to the validation of high-performance liquid chromatography methods based on regulatory guidance documents and accepted industry practices. The information in this chapter gives a brief review of the reasons for performing method validation and the regulations that describe this activity. Individual validation parameters are discussed in relation to the type of method to be validated. Examples of typical validation conditions are presented with references to additional information on individual topics. This chapter was written to help analysts responsible for method validation. 

Kunkel, A., Gunter, S., Dette, C., and Watzig, H. (1997). Quantitation of insulin by capillary electrophoresis and high-performance liquid chromatography — method comparison and validation. J. Chromatogr. A 781, 445—455. 

Shabir, G. A. Validation of high-performance liquid chromatography methods for pharmaceutical analysis. Understanding the differences and similarities between validation requirements of the U S Food and Drug Administration, the US Pharmacopeia and the International Conference on Harmonization. 

Priestner AA. Use of radiolabeled drug substance to investigate mass balance during validation of a high-performance liquid chromatography method for impurities. Anal Proc 1993 30 374-377. 

Cinquina A, Longo F, Anastasi G, et al., Validation of a high performance liquid chromatography method for the determination of oxytetracycline, tetracycline, chlortetracycline and doxycycline in bovine milk and muscle, J. Chromatogr. A 2003 987 227-233. 

Vidovic, S., Stojanovic, B., Veljkovic, J., and Prazic-Arsic, L., 2008. Simultaneous determination of some water-soluble vitamins and preservatives in multivitamin syrup by validated stability-indicating high-performance liquid chromatography method. Journal of Chromatography A. 1202 155-162. 

These authors noted the potential for the assay to underestimate the concentration of TSR due to decreased binding of metabolites relative to parent spinosad. However, the major residue found was parent spinosad, so underestimation of residues is not likely to be problematic. Overall, this method was validated in 34 matrices and showed excellent agreement with results obtained with a high-performance liquid chromatography/ultraviolet detection (HPLC/UV) method. ... 

Full acceptance of HPLC/MS methods by the US EPA OPP as enforcement methods occurred between 1998 and 2001. For example, in 1998, the EPA OPP accepted HPLC/MS (without MS/MS) methods as primary enforcement methods, and high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) only was suitable for confirmatory methods. However, in 2001, HPLC/MS/MS methods also became acceptable for primary enforcement. Table 4 summarizes the types of methods that were validated by the EPA OPP method validation program, for both food tolerance enforcement methods and environmental chemistry methods. 

High performance liquid chromatography-mass spectrometric methods Nitin et al. [75] developed and validated a sensitive and selective liquid chromatography-tandem mass spectrometric method (LC MS MS) for the simultaneous estimation of bulaquine and its metabolites primaquine in monkey plasma. The mobile phase consisted of acetonitrile ammonium acetate buffer (20 mM, pH 6) (50 50, v/v) at a flow rate of 1 mL/min. The chromatographic separations were achieved on two Spheri cyano columns (5 pm, 30 cm x 4.6 mm), connected in... 

Sripalakit, P., Nermhom, P., and Saraphanchotiwitthaya, A. 2006. Validation and pharmacokinetic application of a method for determination of doxazosin in human plasma by high performance liquid chromatography. Biomed Chromatogr. 20 729. 

Wynia, G. S., Windhorst, G., Post, P. C., and Maris, F. A. (1997). Development and validation of a capillary electrophoresis method within a pharmaceutical quality control environment and comparison with high-performance liquid chromatography. /. Chromatogr. A 773(1—2), 339—350. 

T.R.M. De Beer, W.R.G. Baeyens, A. Venneire, D. Broes, J.R Remon, and C. Vervaet, Raman spectroscopic method for the determination of medroxyprogesterone acetate in a pharmaceutical suspension Validation of quantifying abilities, uncertainty assessment and comparison with the high performance liquid chromatography reference method. Anal. Chim. Acta, 589, 192-199 (2007). 

Hansen, A.M., Olsen, I.L.B., Holst, E. and Poulsen, O.M. (1991) Validation of a high-performance liquid chromatography/ fluorescenes detection method for the simultaneous quantification of fifteen polycyclic aromatic hydrocarbons. Annals of Occupational Hygiene, 35, 603-11. 

G. T. Carter, R.E. Schiesswohl, H. Burke, and R. Young, Peak homogeneity determination for the validation of high-performance liquid chromatography assay methods, J. Pharm. Sci., 77 317 (1982). 

Hou W, Watters JW, McLeod HL (2004) Simple and rapid docetaxel assay in human plasma by protein precipitation and high-performance liquid chromatography-tandem mass spectrometry. Journal of Chromatography B 804 263-267 Schuhmacher J, Zimmer D, Tesche F, Pickard V (2003) Matrix effects during analysis of plasma samples by electrospray and atmospheric pressure chemical ionization mass spectrometry practical approaches to their elimination. Rapid Communications in Mass Spectrometry 17 1950-1957 Shah PW (2001) Guidance for Industry Bioanalytical Method Validation U.S. Department of Health and Human Services, Food and Drug Administration... 

Baer et al. during 1986 to 1988. Urine was screened by methods in effect in the federal probation system THC metabolite and opiates were screened by EMIT and confirmed by high performance liquid chromatography (HPLC) at cutoffs of 100 and 500 ng/mL, respectively. Cocaine metabolites and PCP were screened by thin-layer chromatography (TLC) at cutoffs of 2000 and 500 ng/mL, respectively, and confirmed by GC. Hair testing was by RIA. The RIA procedures, validated by GC/MS, showed no false positives due to cross-reactivity effects. 

Validation of high-performance liquid chromatography (HPLC) methods focus mainly on the following ... 

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