An improved size exclusion-HPLC method for molecular size distribution analysis of immunoglobulin G using sodium perchlorate in the eluent.

Size exclusion (SE) high performance liquid chromatography (HPLC) is widely used for the molecular size distribution (MSD) analyses of various therapeutic proteins. We report development and validation of a SE-HPLC method for MSD analyses of immunoglobulin G (IgG) in products using a TSKgel SuperSW3000 column and eluting it with 0.4M NaClO4, a chaotropic salt, in 40mM phosphate buffer, pH 6.8. The chromatograms show distinct peaks of aggregates, tetramer, and two dimers, as well as the monomer and fragment peaks. In addition, the method offers about half the run time (12min), better peak resolution, improved peak shape and more stable base-line compared to HPLC methods reported in the literature, including that in the European Pharmacopeia (EP). A comparison of MSD analysis results between our method and the EP method shows interactions between the protein and the stationary phase and partial adsorption of aggregates and tetramer on the stationary phase, when the latter method is used. Thus, the EP method shows lower percent of aggregates and tetramer than are actually present in the products. In view of the fact that aggregates have been attributed to playing a critical role in adverse reactions due to IgG products, our observation raises a major concern regarding the actual aggregate content in these products since the EP method is widely used for MSD analyses of IgG products. Our method eliminates (or substantially reduces) the interactions between the proteins and stationary phase as well as the adsorption of proteins onto the column. Our results also show that NaClO4 in the eluent is more effective in overcoming the protein/column interactions compared to Arg-HCl, another chaotropic salt. NaClO4 is shown not to affect the molecular size and relative distribution of different molecular forms of IgG. The method validated as per ICH Q2(R1) guideline using IgG products, shows good specificity, accuracy, precision and a linear concentration dependence of peak areas for different molecular forms. In summary, our method gives more reliable results than the SE-HPLC methods for MSD analyses of IgG reported in the literature, including the EP, particularly for aggregates and tetramer. The results are interpreted in terms of ionic (polar) and hydrophobic interactions between the stationary phase and the IgG protein.

Protein Nitrogen Determination by Kjeldahl Digestion and Ion Chromatography.

We report development and validation of a simple, rapid, and accurate method for the quantitation of protein nitrogen, which combines Kjeldahl digestion and ion chromatography with suppressed conductivity detection and requires nanomolar amount of nitrogen in samples (≥10 µg protein). The mechanism of suppressed conductivity detection does not permit analysis of samples containing copper (present in Kjeldahl digestion solution) and aluminum (present in many vaccines as adjuvants) due to precipitation of their hydroxides within the suppressor. We overcame this problem by including 10 µM oxalic acid in Kjeldahl digests and in the eluent (30 mM methanesulfonic acid). The chromatography is performed using an IonPac CS-16 cation exchange column by isocratic elution. The method reduces the digestion time to less than 1 h and eliminates the distillation and titration steps of the Kjeldahl method, thereby reducing the analysis time significantly and improving precision and accuracy. To determine protein nitrogen in samples containing non-protein nitrogen, proteins are precipitated by a mixture of deoxycholate and trichloroacetic acid and the precipitates are analyzed after dissolving in KOH. The method is particularly useful for biological samples that are limited and can also be applied to food, environmental, and other materials.

Development and validation of a stability-indicating high-performance liquid chromatographic assay for ketoprofen topical penetrating gel.

An isocratic RP-HPLC procedure has been developed and validated for the quantitative determination of ketoprofen in a topical gel. The HPLC procedure consist of a YMC ODS-AQ, 5-microm particle size analytical column (150 mm x 4.6 mm); Alltech Econosphere C18, 5-microm particle size guard column; detection at 233 nm; 1 ml/min flow rate; 20-microl injection volume. The mobile phase consisted of pH 3.5 phosphate buffer-water-acetonitrile (8:43:49, v/v). Sample preparation was a simple extraction of ketoprofen with mobile phase. The above conditions resolved and eluted ketoprofen, excipients, and potential degradants within 35 min, with ketoprofen eluting at about 6.5 min. The procedure was validated with respect to specificity, accuracy, precision, and linearity. The accuracy of the procedure, determined by spike recovery measurements, was 100.1-100.5%. The intra- and inter-day precisions were demonstrated by the relative standard deviations (RSD) of 0.3-0.6% and 0.5%, respectively. The intermediate precision was determined by comparing the results obtained with four independently prepared samples by two chemists using two columns on different days. The results indicate no significant difference (P = 0.17). The procedure showed linearity over the concentration range 4 x 10(-5) to 1 x 10(-1) mg/ml.

A stability-indicating HPLC assay for metronidazole benzoate.

A simple and rapid stability-indicating HPLC assay procedure has been developed and validated for metronidazole benzoate. The HPLC conditions were as follows, column: Waters Symmetry C8, 5 microm packing, 4.6 mm x 250 mm; detection: UV at 271 nm; injection volume: 20 microl; mobile phase: acetonitrile-0.1% glacial acetic acid in monobasic potassium phosphate (0.01 M) (40:60, v/v); isocratic elution under ambient temperature at 2.0 ml min(-1). The procedure separated metronidazole benzoate and its potential degradation products, metronidazole and benzoic acid, in an overall analysis time of about 6 min with metronidazole benzoate eluting at about 5 min. The injection repeatability was 0.03%, and the intraday and interday repeatability were 0.4 and 0.7%, respectively. The procedure provided a linear response over the concentration range 0.2-800 microg ml(-1) (r=1.0000) with the limits of detection and quantitation 0.03 and 0.2 microg ml(-1), respectively. The solubilities of metronidazole benzoate in water, 0.01 M hydrochloric acid and 0.05 M phosphate buffer, pH 6.8, determined each in triplicate using the procedure, were 0.2 mg ml(-1) (R.S.D. 7%), 0.4 mg ml(-1) (R.S.D. 2%) and 0.2 mg ml(-1) (R.S.D. 8%), respectively. The results show no detectable hydrolysis of metronidazole benzoate in 0.01 M hydrochloric acid at 37 degrees C or in the mobile phase at ambient temperature in 10 h.

Development and validation of an assay for citric acid/citrate and phosphate in pharmaceutical dosage forms using ion chromatography with suppressed conductivity detection.

The paper describes the development and validation of a simple, rapid, accurate, and sensitive ion chromatographic procedure to assay total citrate (citric acid/citrate) and phosphate in nine dosage forms. The dosage forms chosen represent all dosage forms in USP27-NF22 for which the respective monographs require an assay for either citric acid/citrate or citric acid/citrate and phosphate. Citrate and phosphate were separated in <10min by a hydroxide-selective column using anion-exchange chromatography with a 20mM potassium hydroxide eluent and detected by suppressed conductivity. The method showed linear responses over the concentration ranges 0.2-100microg ml(-1) (r(2) > 0.9990) for citrate and 0.2-60microg ml(-1) (r(2) = 0.9999) for phosphate, with limits of quantitation (signal-to-noise (S/N) = 10) of 0.2microg ml(-1) for both analytes. The accuracy of the procedure, determined by spiked recovery measurements, was within 95-105%. The intraday and the interday precision were demonstrated by the relative standard deviations (R.S.D.) of <1 and <2%, respectively, for both analytes. The ruggedness was determined by a full factorial design using analyst, equipment, column lot, and eluent preparation procedure as variables. The results show an overall R.S.D. of <3% and that an electrolytically generated 20mM KOH eluent produces assay results equivalent to a manually prepared 20mM NaOH eluent.

Strategy for streamlined release identity testing of chromatography media.

In accordance with the US Code of Federal Regulations 21CFR 211.84 (6)(d)(1), a specific identity test must be performed for the release of chromatography media (stationary phase) before use in production of human pharmaceuticals. Due to the complexity of the physical and chemical properties of these media, i.e., variable particle morphology, insolubility, and chemical inertness, the development of specific identity tests presents a challenge. In this paper we report a new strategy for media identification that uses a combination of three relatively simple techniques: Fourier transform infrared (FT-IR) and near infrared (NIR) spectroscopy in conjunction with search libraries, and particle size distribution analysis. The methods are well established and suitable for routine application in a quality control laboratory. A hierarchical selection procedure utilizing these methods permits assignment of a unique identity for each of the chromatography media in use at a given facility, and form the basis of release tests for the media. Although this strategy was developed using specific media, the generic nature of the technology and the selection strategy proposed would permit its application to other chromatography media as well.