Choosing an equivalent replacement column for a reversed-phase liquid chromatographic assay procedure

The column selectivity parameters (H, S*, A, B and C) described in the preceding paper [L.R. Snyder, A. Maule, A. Heebsch, R. Cuellar, S. Paulson, J. Carrano, L. Wrisley C.C. Chan, N. Pearson, J.W. Dolan, J.J. Gilroy, J. Chromatogr. A 1057 (2004) 49-57] can be used to compare columns in terms of selectivity. A detailed procedure for such column comparisons is presented here, and evaluated by its use in finding suitable replacement columns for 12 different routine separations performed in five different pharmaceutical analysis laboratories.     

Development of an electrochemical detector for high-performance liquid chromatographic assay of brain catecholamines

An electrochemical detector that utilizes the reduction-oxidation phenomena of electrochemically active substances has been developed for high-performance liquid chromatography. The construction and application of the detector to brain catecholamines, particularly for norepinephrine and dopamine analysis, are described. The differences between this detector and those reported previously are discussed.     

Robustness testing of a reversed-phase high-performance liquid chromatographic assay: comparison of fractional and asymmetrical factorial designs

Robustness tests were performed on a reversed-phase HPLC assay for triadimenol. Different experimental designs were compared. Two-level fractional factorial designs with different resolutions were used to study the influence of procedure-related factors. The factors chromatographic column manufacturer at four levels and instrument at three levels were stepwise included in the study using asymmetrical factorial designs. The significance of the factor effects was determined statistically, using two types of error estimates in the calculation of critical effects, and graphically, by means of half-normal plots. The asymmetrical designs turned out to be an efficient and economic method to examine the influence of factors at different numbers of levels in the robustness testing of analytical methods.     

Development and use of a stability-indicating high-performance liquid chromatographic assay for Meldrum's acid.

A high-performance liquid chromatographic method was developed that separates Meldrum's acid from its primary decomposition products, malonic acid and acetone. The method uses a reversed-phase column under isocratic conditions, with detection by ultraviolet absorption at 210 nm. Quantitation of the parent molecule and the acid decomposition product was possible over concentration ranges of 0.1-10.0 and 0.1-2.0 mg/ml, respectively. Acetone could be determined only at much higher concentrations. Using the malonic acid concentration as a measure of decomposition, this method was used to determine the hydrolytic stability of Meldrum's acid and its skin penetration properties.     

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.     

Silica versus polymer-based stationary phases for reversed-phase high-performance liquid chromatographic analyses of rat insulin biosynthesis. A comparison of resolution and recovery

Because of the problems caused by the irreversible binding of insulins and proinsulins to several silica-based reversed-phase columns, the use of polymeric reversed-phase columns was investigated for the analysis of rat islet polypeptides involved in insulin biosynthesis. No irreversible binding of insulins and proinsulins was observed for the polymeric reversed-phase columns, probably due to the absence of silanol groups. The six polypeptides involved in insulin biosynthesis in rat islets were equally well resolved in shallow trifluoroacetic acid-acetonitrile gradients on the silica-based Nucleosil 300-5C4 column (45 degrees C), the polymer-based Asahipak C4P-50 (25 and 45 degrees C), and ODP-50 columns (45 degrees C). In shallow triethylammonium phosphate-acetonitrile gradients (25 degrees C) satisfactory resolution of the two rat proinsulins was only obtained on the polymer-based Asahipak C4P-50 and C8P-50 columns. Increasing the separation temperature to 45 degrees C improved the separation of the two insulins and the two proinsulins in all cases. The shifts in retention times for the individual islet polypeptides observed in relation to the increased separation temperature were found to be different for the silica C4 and the polymer C4 columns. Recoveries of rat islet polypeptides were comparably high from the silica- and the polymer-based C4 columns and linear load-response curves were obtained in the microgram to picogram mass range on both columns.     

Development and validation of a reversed-phase ultra-performance liquid chromatographic method for assay of lacidipine and related substances

A simple isocratic, RP-ultra-performance LC method was developed and validated for the determination of lacidipine, three process impurities formed during synthesis, and three degradation products present in drug substance and the drug product. An efficient chromatographic separation was achieved on an Acquity BEH C18 column using pH 4.5 ammonium acetate-acetic acid buffer-methanol (70 + 30, v/v) mobile phase. The monitoring wavelength was 240 nm, and the flow rate 0.25 mL/min. Forced degradation studies using acid, alkali, peroxide, water, heat, and light were conducted, and all impurities were separated. The method was validated successfully for specificity, precision, linearity, accuracy, LOD, LOQ, and robustness, according to International Conference on Harmonization guidelines. The linearity of the calibration curve for lacidipine and each impurity was found to be very good (r2 > 0.999). This method is shown to be suitable for analysis of lacidipine to evaluate the quality of drug substance and a drug product.     

High-performance liquid chromatographic stability indicating assay method of tianeptine sodium with simultaneous fluorescence and UV detection

The purpose of this work is to develop a sensitive, selective, and validated stability-indicating HPLC assay of tianeptine (TIA) in bulk drug and tablet form. TIA is subjected to different stress conditions, including UV-light, oxidation, acid base-base hydrolysis, and temperature. TIA and its possible degradation products are analyzed on Agilent-Zorbax-XDB-C18 column using gradient elution with acetonitrile and 0.02M sodium acetate (pH 4.2). The samples are monitored simultaneously with photo-diode array at 254 nm and fluoroscence detector set to 350 nm (ex) and 425 nm (em). TIA is integrated from its UV-chromatogram, and the photodecomposition products are integrated from the fluoroscence-chromatogram. TIA and its photodecomposition products are separated by TLC using ethyl acetate-n-hexane-glacial acetic acid-methanol (10.0:14.0:0.2:1.0, v/v) as developing system. One potential photodegradation product is detected by fluoroscence in TIA-tablet form and separated by TLC. The linear range of TIA is between 0.5 to 50 microg/injection with limits of quantitation and detection values of 30 and 8 ng/injection, respectively. The inter-assay percentage of deviation is not more than 0.03%, and the day-to-day variation is not more than 0.1%.     

Validation of a solid-phase extraction high-performance liquid chromatographic assay for doxazosin.

The determination of doxazosin by high-performance liquid chromatography with fluorescence detection is described. Propanolol was used as the internal standard. Plasma samples were treated with methanol to precipitate the proteins. Doxazosin was isolated with C18 reversed-phase extraction columns. The determination limit is 1 ng/ml of plasma, while the extraction columns can be reused frequently. The method is applied to clinical trial samples.     

Reversed-phase high-performance liquid chromatographic method for the assay of oxytetracycline

The British Pharmacopoeia monograph for oxytetracycline calcium describes an high-performance liquid chromatographic (HPLC) assay which requires packing of the column by the analyst. Presented in this report is an HPLC method for the assay of oxytetracycline which employs a commercially available reversed-phase column and a solvent system which gives improved separation of the antibiotic from common impurities. Results obtained using this method for both bulk and dosage forms of oxytetracycline are in accord with the results of the microbiological assays.     

Development and validation of a reversed-phase ion-pair high-performance liquid chromatographic method for the determination of risedronate in pharmaceutical preparations

A stability indicating, reversed-phase ion-pair high-performance liquid chromatographic method was developed and validated for the determination of risedronate in pharmaceutical dosage forms. The determination was performed on a BDS C₁₈ analytical column (250 mm × 4.6 mm i.d., 5 μm particle size); the mobile phase consisted of 0.005 M tetrabutylammonium hydroxide and 0.005 M pyrophosphate sodium (pH 7.0) mixed with acetonitrile in a ratio (78:22, v/v) and pumped at a flow rate 1.00 mL min⁻¹. The ultraviolet (UV) detector was operated at 262 nm. The retention times of magnesium ascorbyl phosphate, which was used as internal standard and risedronate were 4.94 and 5.95 min, respectively. The calibration graph was ranged from 2.50 to 20.00 μg mL⁻¹, while detection and quantitation limits were found to be 0.48 and 1.61 μg mL⁻¹, respectively. The intra- and inter-day percentage relative standard deviations, %R.S.D., were less than 5.9%, while the relative percentage error, %E_r, was less than 0.4%. The method was applied to the quality control of commercial tablets and content uniformity test and proved to be suitable for rapid and reliable quality control.     

Chemically bonded phases for the reversed-phase high-performance liquid chromatographic separation of basic substances

A chemically bonded phase with a peptide group (PB) for reversed-phase high-performance liquid chromatography (HPLC) is described. This packing was prepared by a two-stage modification of the surface of silica gel with mono- and trifunctional 3-aminopropylsilane and then with an appropriate derivative of a fatty acid. Packings prepared in this way were compared with standard C18 materials used in HPLC. Surface characteristics of the packings before and after chemical modification were determined by different physico-chemical methods, e.g., porosimetry, elemental analysis, 13C and 29Si cross-polarization magic angle spinning NMR and HPLC. Chromatographic properties of these packings were evaluated by comparison between log k' of one phase and log k' of a second phase for substances with different chemical natures. The PB packing was found to be especially useful for the separation of basic substances.     

Development of a new assay for the screening of hypochlorous acid scavengers based on reversed-phase high-performance liquid chromatography

A new assay for the screening of hypochlorite/hypochlorous acid (XOCl) scavengers, based on the reversed-phase high performance liquid chromatographic analysis of human serum albumin (HSA, 0.2% in 100 mM sodium phosphate, pH 7), before and after oxidation by XOCl (1.6 mM), was developed. XOCl induced a significant decrease of the area under the chromatographic peak of HSA at 280 nm due to the oxidation of the aromatic amino acids tryptophan and tyrosine, as suggested by the literature and by the chromatographic analyses and the electrochemical study performed here. The assay was validated by testing known XOCl scavengers such as ascorbic acid, cysteine, glutathione, S-methylglutathione and alpha-lipoic acid and other antioxidants such as carnosine and chlorogenic acid, which inhibited the oxidation of HSA. Quantitative activities were calculated using an original formula based on the changes of the area of the albumin peak. Electrochemical data collected here in a homogeneous medium showed that the anodic potentials of the antioxidants tested are less positive (ascorbic acid, chlorogenic acid and cysteine) or similar (alpha-lipoic acid) compared with those of the aromatic residues (tryptophan and tyrosine) of HSA oxidized by XOCl. However, as expected, carnosine, glutathione and S-methylglutathione were inactive at a glassy-carbon, gold or platinum electrode.     

Comparison of reversed-phase and weak anion-exchange high-performance liquid chromatographic methods for peptide separations

Weak anion-exchange and reversed-phase high-performance liquid chromatographic methods for peptide separations were compared using a tryptic digest of "rat small myelin basic protein". In these experiments, a number of tryptic peptides that were not resolved on the reversed-phase column could be separated on the weak anion-exchange column, and in other instances, as might be expected, reversed-phase chromatography provided better resolution of certain peptides than did the weak anion-exchange method. The results obtained strongly suggest that the combined use of these two methods of separation, which utilize different selectivities, can provide an excellent improvement in resolving power for a number of peptide separations.     

Stability indicating reversed-phase high-performance liquid chromatographic and thin layer densitometric methods for the determination of ziprasidone in bulk powder and in pharmaceutical formulations

Two sensitive and reproducible methods were developed and validated for the determination of ziprasidone (ZIP) in the presence of its degradation products in pure form and in pharmaceutical formulations. The fi rst method was based on reversed-phase high-performance liquid chromatography (HPLC), on a Lichrosorb RP C(18) column using water:acetonitrile:phosphoric acid (76:24:0.5 v/v/v) as the mobile phase at a fl ow rate of 1.5 mL min(-1) at ambient temperature. Quantification was achieved with UV detection at 229 nm over a concentration range of 10-500 micro g mL(-1) with mean percentage recovery of 99.71 +/- 0.55. The method retained its accuracy in presence of up to 90% of ZIP degradation products. The second method was based on TLC separation of ZIP from its degradation products followed by densitometric measurement of the intact drug spot at 247 nm. The separation was carried out on aluminium sheet of silica gel 60 F(254) using choloroform:methanol:glacial acetic acid (75:5:4.5 v/v/v) as the mobile phase, over a concentration range of 1-10 micro g per spot and mean percentage recovery of 99.26 +/- 0.39. Both methods were applied successfully to laboratory prepared mixtures and pharmaceutical capsules.