Validation of liquid chromatographic methods

J. Fogel, J. Sisco, and F. Hess, Validation of liquid chromatographic method for assay of chlorthalidone in tablet formulations, J. Assoc. Off. Anal. Chem., 68 96 (1985). 

Clark, M.J.R. Whitfield, P.H. Conflicting perspectives about detection hmits and about the censoring of environmental data. Water Resour. BuU. 1994, 30, 1063 1079. Jenke, D.R. Poss, M. Story, J. Odufu, A. Zietlow, D. Tsilpetros, T. Development and validation of liquid chromatographic methods for the identification and quantification of organic compounds leached from a laminated polyolefin material. J. Chromatogr. Sci. in press. Jenke, D.R. Chromatographic method vahdation A review of current practices and procedures Part II. Guidelines for primary validation parameters. Instrum. Sci. Technol. 1998, 26, 1-18. 

The focus of this chapter is validation of liquid chromatographic methods. Numerous articles describe details and provide examples for LC method validation [6,7] therefore, a step-by-step guide to method validation is not provided here. This chapter provides an overview of LC method validation as well as a discussion about some of the pitfalls associated with traditional validation and some alternative practices that are currently being discussed in the literature. 

Rao Nageswara, R., Nagaraju, D., Parimala, P. and Adapa, R. S. Development and validation of liquid chromatographic method for monitoring reaction involved in the synthesis of 4-methoxyphenylacetic acid. J. Chromatogr. A 972 277-282, 2002. 

Schubert et al. [72] developed and validated a liquid chromatographic method for the determination of omeprazole in powder for injection and in pellets. The analyses were performed at room temperature on a reversed-phase Ci8 column of 250 mm x 4.6 mm (5 /im). The mobile phase, composed of methanol-water (90 10) was pumped at a constant flow-rate of 1.5 ml/min. Detection was performed on a UV detector at 301 nm. The method was validated in terms of linearity, precision, accuracy, and ruggedness. 

Tab. 2.4. Liquid-liquid chromatographic methods for measurement of log D at pH 7.4. Results for a series of validation compounds.

Gagliardi et al. [72] developed a simple high performance liquid chromatographic method for the determination of miconazole and other antimycotics in cosmetic antidandruff formulations. This high performance liquid chromatographic method was carried out on a Discovery RP Amide Ci6 column and spectrophotometric detection was performed at 220 nm. The initial mobile phase was a mixture of acetonitrile and aqueous 0.001 M sodium perchlorate (pH 3) in the ratio of 15 85 (v/ v) then a linear gradient less than 46% acetonitrile in 70 min, and less than 50% in 80 min. The extraction procedure was validated by analyzing samples of shampoo... 

Gupta, M. and Bhargava, H. N., Development and validation of a high-performance liquid chromatographic method for the analysis of bndesonide. Journal of Pharmaceutical and Biomedical Analysis 40(2), 423-428, 2006. 

PQ should be performed on a daily basis or whenever the instrument is used. The test frequency not only depends on the stability of the equipment but on everything in the system that may contribute to the results. For a liquid chromatograph, this may be the chromatographic column or a detector s lamp. The test criteria and frequency should be determined during the development and validation of the analytical method. 

There are no official guidelines on the sequence of validation experiments, and the optimal sequence can depend on the method itself. For a liquid chromatographic method the sequence from Table 2 has been proven to be useful. 

Figure 11.5 Chromatograms of plasma samples obtained with fully automated on-line SPE-LC (a) drug-free human plasma (b) human plasma spiked with omeprazole (100 ng/ml) and phenacetin (internal standard 1000 ng/ml). Reprinted from Journal of Pharmaceutical and Biomedical Analysis, 21, G. Garcia-Encina et al., Validation of an automated liquid chromatographic method for omeprazole in human plasma using on-line solid-phase extraction, pp. 371-382, copyright 1999, with permission from Elsevier Science.

Castro et al. [64] reported a comparison between derivative spectro-photometric and liquid chromatographic methods for the determination of omeprazole in aqueous solutions during stability studies. The first derivative procedure was based on the linear relationship between the omeprazole concentration and the first derivative amplitude at 313 nm. The first derivative spectra were developed between 200 and 400 nm (A/ = 8). This method was validated and compared with the official HPLC method of the USP. It showed good linearity in the range of concentration studied (10—30 /ig/ ml), precision (repeatability and interday reproducibility), recovery, and specificity in stability studies. It also seemed to be 2.59 times more sensitive than the HPLC method. These results allowed to consider this procedure as useful for rapid analysis of omeprazole in stability studies since there was no interference with its decomposition products. 

High-performance liquid chromatographic methods are the most common form of analytical technique used to support drug product registration. An example of a validation protocol for an HPLC assay and related compounds method is provided in Example 2. 

Woo JS (2006) Nasal absorption studies of granisetron in rats using a validated high-performance liquid chromatographic method with mass spectrometric detection. Arch Pharm Res 30 778-784... 

Nygaard L, Kilde HD, Andersen SG, Henriksen L, Overby VJ. 2004. Development and validation of a reversed-phase liquid chromatographic method for analysis of degradation products of estradiol in Vagifem tablets. J. Pharm. Biomed. Anal. 34 265 - 276. 

Manna et al. described a rapid and accurate reversed-phase ion-paring liquid chromatographic method, with ultraviolet detection, for the simultaneous assay of benazepril hydrochloride, fosinopril sodium, ramipril and hydrochlorothiazide [32], The separation was achieved on a LC-8 (12.5 cm x 4 mm, 5 pm particle size) column. The mobile phase consisted of 20 mM sodium heptansulfonate (pH 2.5) and methanol (8 17 v/v). Validation of the method showed it to be precise, accurate, and linear over the concentration range of analysis, with a limit of detection of 2 ng for benazepril. The method was applied to the analysis of three different binary commercial formulations. 

Gana et al. developed and validated a reversed-phase high performance liquid chromatographic method for the kinetic investigation of the chemical and enzymatic hydrolysis of benazepril hydrochloride [37]. Kinetic studies on the acidic hydrolysis of benazepril hydrochloride were carried out in 0.1 M hydrochloric acid solution at 50, 53, 58 and 63°C. Benazepril hydrochloride appeared stable in pH 7.4 phosphate buffer at 37°C, and showed susceptibility to in vitro enzymatic hydrolysis with porcine liver esterase (PLE) in a pH 7.4 buffered solution at 37°C. 

The assay of an IV formulation is performed using a liquid chromatographic method, which requires the following solutions. For solution (1), dilute a quantity of the intravenous infusion with sufficient mobile phase to produce a solution containing the equivalent of 0.05% w/v of ciprofloxacin. Solution (2) contains 0.058% w/v of ciprofloxacin hydrochloride EPCRS in mobile phase. Solution (3) contains 0.025% w/v of ciprofloxacin impurity C EPCRS (ethylenediamine compound) in solution (2). For solution (4), dilute 1 volume of solution (3) to 100 volumes with the mobile phase. The chromatographic procedure may be carried out using a stainless steel column (12.5 cm x 4 mm) packed with stationary phase C (5 mm) (Nucleosil C18 is suitable). The mobile phase is eluted at a flow rate of 1.5 mL/min, and consists of a mixture of 13 volumes of acetonitrile and 87 volumes of a 0.245% w/v solution of orthophosphoric acid (the pH of which has been adjusted to 3.0 with triethylamine). Analytes are detected on the basis of their UV absorption at 278 nm. The assay is not valid unless in the chromatogram obtained with solution (3), the resolution factor between the peaks due to ciprofloxacin and the ciprofloxacin impurity C is at least 1.5. 

Gioia, M.G. et al. Development and validation of a liquid chromatographic method for the determination of ascorbic acid, dehydroascorbic acid and acetaminophen in pharmaceuticals. J. Pharm. Biomed. 2008, 48, 331-339. 

Kyriakides, D. and Panderi 1. Development and validation of a reversed-phase ion-pair high-performance liquid chromatographic method for the determination of risedronate in pharmaceutical preparations. Anal. Chim. Acta 2007,584,153-159. 

R. K. Verma, S. Garg, A validated high performance liquid chromatographic method for analysis of isosorbide mononitrate in bulk material and extended release formulations, /. Pharm. Biomed. Anal. 30 (2002), 583-591. 

M. J. Nozal, J. L. Bernal, J. J. Jimenez, M. T. Martin, and F. J. Diez, Development and validation of a liquid chromatographic method for determination of lacidip-ine residues on surfaces in the manufacture of pharmaceuticals, /. Chromatogr. A... 

Arun et al. [30] developed and validated a rapid high-performance liquid chromatographic method for the estimation of buclizine hydrochloride in tablet dosage form. The stationary phase used was precoated silica gel 60 F254. The mobile phase used was a mixture of methanol chloroform ammonia (8 1 1). The detection of spots was carried out at 234 nm. The method was validated in terms of linearity, accuracy, precision, and specificity. The calibration curve was linear between 100 and 700 ng/spot. The limit of detection and the limit of quantification were 20 and 100 ng/spot, respectively. The method can be used to determine the drug content of tablet dosage formulation. 

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