Development and validation of an improved liquid chromatographic method for the analysis of oxytetracycline

Summary An improved LC method is described for the separation of oxytetracycline and its impurities. The separation is much better than that obtained with official pharmacopoeia methods. The method uses XTerra RP-18, 5 μm (25cm×4.6 mm I.D.), a silica-based stationary phase with methyl end-capping, claimed to reduce silanol activity. The column temperature is set at 30°C and a UV detection is performed at 280 nm. Mobile phase containing acetonitrile −0.25 M tetrabutylammonium hydrogen sulfate pH 7.5−0.25 M ethylenediaminetetraacetic acid pH 7.5-water (115:360:160:365,v/v/v/v) is used at a flow rate of 1.0 mL.min⁻¹, to separate the impurities present in oxytetracycline base. A central composite experimental design is used to optimize the separation. A second isocratic method with higher content of acetonitrile is needed to separate the more retained impurities present only in oxytetracycline hydrochloride. The method is robust and shows good selectivity, repeatability, linearity and sensitivity.     

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.     

Determination, purity assessment and chiral separation of levodopa methyl ester in bulk and formulation pharmaceuticals

A sensitive, reliable and reproducible HPLC method with electrochemical detection (HPLC-ECD) has been developed for the separation and quantification of levodopa methyl ester (LDME) and its impurities such as levodopa (l-DOPA), 3-methoxytyrosine (MTS) and l-tyrosine (TS) in bulk drug and pharmaceutical dosage form. The separation was performed on an LC18 column by isocratic elution with methanol-acetonitrile-50 mm potassium dihydrogen phosphate (8:2:90, v/v/v) containing 5 mm sodium 1-hexanesulfonate, 5 mm EDTA and 5 mm sodium chloride, adjusted with phosphoric acid to a pH of 3.2 as mobile phase. The correlation coefficients of linear regression for LDME, L-DOPA, MTS and TS were more than 0.999. The detection limits for L-DOPA, MTS and TS were 3.15, 2.04 and 2.88 ng/mL, respectively. The precision was checked in terms of F-test variance ratio using potentiometric titration as reference. The separation of dopa methyl ester enantiomers by chiral chromatography is also described. This method is capable of separating the two enantiomers with a selection of 1.4 and a resolution of 8.4. Both methods were found to be stable and useful in the quality control of the bulk material and formulations.     

A Stability-Indicating LC Method for Rimonabant

A simple, inexpensive and rapid isocratic LC method has been developed for the quantative determination of Rimonabant, an anti-obesity drug. The method can also be employed for the determination of Rimonabant and its impurities in the bulk drug. Degradation studies were performed on the bulk drug by heating to 60 °C, exposure to UV light at 254 nm, acid (0.5 N hydrochloric acid), base (0.5 N sodium hydroxide) and aqueous hydrolysis and oxidation with 3.0% v/v hydrogen peroxide. Considerable degradation was observed under oxidation conditions. Good resolution between the peaks corresponding to impurities produced during synthesis, degradation products and the analyte was achieved on a Phenomenex Gemini C18 LC column using a mobile phase consisting of a mixture of aqueous potassium dihydrogen phosphate (pH 3.0) and acetonitrile. The degradation samples were assayed against the reference standard of Rimonabant and the mass balance in each case was close to 99.5%. Validation of the method was carried out as per ICH requirements.     

Enantiomeric separation of amlodipine and its two chiral impurities by nano‐liquid chromatography and capillary electrochromatography using a chiral stationary phase based on cellulose tris(4‐chloro‐3‐methylphenylcarbamate)

In this work, a novel polysaccharide‐based chiral stationary phase, cellulose tris(4‐chloro‐3‐methylphenylcarbamate), also called Sepapak 4 has been evaluated for the chiral separation of amlodipine (AML) and its two impurities. AML is a powerful vasodilatator drug used for the treatment of hypertension. Capillary columns of 100 μm id packed with the chiral stationary phase were used for both nano‐LC and CEC experiments. The optimization of the mobile phase composed of ACN/water, (90:10, v/v) containing 15 mM ammonium borate pH 10.0 in nano‐LC allowed the chiral separation of AML and the two impurities, but not in a single run. With the purpose to obtain the separation of the three pairs of enantiomers simultaneously, CEC analyses were performed in the same conditions achieving better enantioresolution and higher separation efficiencies for each compound. To fully resolve the mixture of six enantiomers, parameters such as buffer pH and concentration sample injection have been then investigated. A mixture of ACN/water (90:10, v/v) containing 5 mM ammonium borate buffer pH 9.0 enabled the complete separation of the three couples of enantiomers in less than 30 min. The optimized CEC method was therefore validated and applied to the analysis of pharmaceutical formulation declared to contain only AML racemate.     

Liquid chromatographic method for analysis of saponins in Maesa balansae extract active against leishmaniasis

A liquid chromatographic method was developed for the separation of six related triterpenoid saponins in Maesa balansae extracts with different purity, active against leishmaniasis. As stationary phase a Hypersil BDS C18 column (3 microm), 100 x 4.6 mm was used. The mobile phase was a mixture of methanol, acetonitrile, 5% (m/v) ammonium acetate, pH 6.5 and water. A linear gradient was developed for the analysis of crude extracts. An isocratic method was developed to analyze purified samples that mainly contained saponins 3 and 4, the most active saponins. The isocratic LC method was optimized and the robustness was evaluated with an experimental design. The method showed good selectivity, repeatability, linearity and sensitivity.     

Isocratic liquid chromatographic method for the analysis of azithromycin and its structurally related substances in bulk samples

An isocratic liquid chromatographic method with UV detection at 215 nm, which is suitable for the analysis of azithromycin (AZT) in bulk samples, is described. AZT is separated from its synthesis intermediates and a degradation product as well as from six unknown impurities on an XTerra RP18 column at 70 degrees C using a mobile phase consisting of acetonitrile-pH 6.5 0.2M K2HPO4-water (35:10:55, v/v/v) at 1.0 mL/min. The XTerra stationary phase contains methyl groups that are incorporated in the bulk structure of the material. This allows for special selectivities. Robustness is evaluated by a full factorial design experiment. The method shows good selectivity, repeatability, linearity, and sensitivity.     

Identification of impurities in polymyxin B and colistin bulk sample using liquid chromatography coupled to mass spectrometry

The European Pharmacopoeia (Ph. Eur.) describes liquid chromatography-ultraviolet (LC-UV) methods using C₁₈ stationary phases for the analysis of polymyxin B and colistin.Several unknown impurities were detected in commercial samples of those polypeptide complexes. However, the Ph. Eur. does not specify any related substances for polymyxin B and colistin. Since both methods use non-volatile buffers, the mobile phases were incompatible with mass spectrometry (MS). For the identification of related substances in bulk samples by LC/MS, volatile mobile phase systems were developed. However, the LC/MS methods (with volatile additives) showed inferior chromatographic separation compared to the LC-UV method (with non-volatile additives). Moreover, previously identified impurities by LC/MS could not be assigned in LC-UV methods as the separation in both systems was different.In this study, known impurities were traced in the LC-UV methods and new impurities present in polymyxin B and colistin bulk samples were characterized. To achieve this, each peak from the non-volatile system was collected separately and reinjected into an LC system with a volatile mobile phase coupled to MS. This way, collected impurity peaks were rechromatographed on a reversed phase column in order to separate the analyte from the buffer salts. Using this method, out of 39 peaks, five novel related substances were characterized in a polymyxin B bulk sample. Fourteen impurities, which were already reported in the literature were traced as good as possible in the LC-UV method. In the case of colistin, a total of 36 peaks were investigated, among which four new compounds. Additionally, 30 known impurities were traced in the LC-UV method.     

Electrophoretron: a new method for enhancing resolution in electrokinetic separations

Until now no liquid chromatography (LC) method is described to determine the purity and content of troleandomycin and its related substances. A simple, robust, sensitive and selective isocratic liquid chromatographic method suitable for the determination of the antibiotic troleandomycin and its related substances is described. This method utilizes as a stationary phase: XTerra RP18 5 microm (25 cm x 4.6 mm I.D.) at 30 degrees C and as mobile phase: acetonitrile-0.2 M ammonium acetate buffer (pH 6.0)-water (45:5:50, v/v), delivered at a flow-rate of 1.0 ml/min. UV detection is performed at 205 nm. Troleandomycin is separated from the partially acetylated related substances and from several unknown impurities present in commercial samples. The robustness of the method was evaluated by a full-factorial experimental design.     

Development of a New Analytical Method for Determination of Related Components in Nateglinide

An isocratic reverse phase liquid chromatographic (RP-LC) assay method has been developed for the quantitative determination of nateglinide and its related components namely imp-1 and imp-2 in bulk drug and in pharmaceutical dosage form, used for the treatment of type II diabetes mellitus. The developed method is stability indicating and also can be used for stability testing. The chromatographic separation was achieved on C-8, 150 × 4.6 mm, 3.5 μm stationary phase. The LC method employs solution A as mobile phase. Solution A contains a mixture of phosphate buffer pH 3.0: acetonitrile (50:50 v/v). The flow rate was 1.0 mL min⁻¹ and the detection wavelength was 210 nm. In the developed LC method the resolution between nateglinide and its potential impurities namely imp-1 and imp-2 was found to be greater than 5.0. The drug was subjected to stress conditions of hydrolysis, oxidation, photolysis and thermal degradation. Considerable degradation was found to occur in acid medium, alkaline medium and oxidative stress conditions. The stress samples were assayed against a qualified reference standard and the mass balance was found close to 99.2%. The developed RP-LC method was validated with respect to linearity, accuracy, precision and robustness.     

Improved High Performance Liquid Chromatography of Cyclic Polypeptide Antibiotics—Polymyxins B—and Its Application to Assays of Pharmaceutical Formulations

Abstract

An isocratic high performance liquid chromatographic (HPLC) method for the analysis of polymyxins B1 and B2 is described. The method uses a 25 cm Hypersil—ODS column, a mobile phase containing 22.5% acetonitrile (v/v) in an aqueous phase with tetramethylammonium chloride (TMAC), a flow rate of 1.09 ml/minute and a wavelength of 220 nm for detection. Complete resolution of B1 and B2, and their separation from all other components and/or impurities have been achieved in less than 23 minutes. The capability of the method for the determination of the polymyxin content in pharmaceutical preparations has also been demonstrated.     

Isocratic separation of erythromycin, related substances and degradation products by liquid chromatography on XTerra RP18

Summary A simple, sensitive, selective and robust isocratic LC method is described for the analysis of erythromycin on XTerra RP₁₈. The main component, erythromycin A, is separated from all known related substances and degradation products. Several unknown impurities are also separated. Acetonitrile-0.2 MK₂HPO₄pH7.0-water, (35∶5∶60, v/v) was used as a mobile phase at 1.0 mL min⁻¹. UV detection was at 215 nm. The robustness of the method was evaluated by a full-factorial experimental design.     

Development and validation of a reversed-phase HPLC method for monitoring of synthetic reactions during the manufacture of a key intermediate of an anti-hypertensive drug

A reversed-phase high-performance liquid-chromatographic method for monitoring of reactions involved in process development of a key intermediate of antihypertensive drugs, e.g, doxazosin mesylate, prazosin, alfuzosin, terazosin, etc., has been developed and validated. The HPLC profiles of impurities of 4-amino-2-chloro-6,7-dimethoxyquinazoline were used as fingerprints to follow the synthetic procedures in the manufacturing unit. The separation was accomplished on an Inertsil ODS-3 column with isocratic elution using acetonitrile-ammonium acetate (10 mM; pH 4.0; 50:50 v/v) as mobile phase and a photodiode array detector set at 240 nm at ambient temperature. The method was validated with respect to accuracy, precision, linearity, and limits of detection and quantification. The method could detect the impurities at a level of 0.01 to 0.20 microg/mL and it was found to be suitable not only for monitoring of reactions but also for quality assurance of 4-amino-2-chloro-6,7-dimethoxyquinazoline.     

Development and Validation of a New Analytical Method for the Determination of Related Components in Quetiapine Hemifumarate

A simple, sensitive isocratic rapid resolution liquid chromatographic assay method has been developed for the quantitative determination of quetiapine hemifumarate in bulk active pharmaceutical ingredient, used for the treatment of schizophrenia. The developed method is also applicable for the process related impurities determination. Efficient chromatographic separation was achieved on a C18 stationary phase with simple mobile phase combination delivered in a isocratic mode and quantification was by ultraviolet detection at 225 nm at a flow rate of 1.0 mL min^?1. In the developed LC method the resolution between quetiapine hemifumarate and its three potential impurities was found to be greater than 2.0. Regression analysis showed an r value (correlation coefficient) greater than 0.99 for quetiapine hemifumarate and its three impurities. This method was capable to detect all three impurities of quetiapine hemifumarate at a level of 0.003% with respect to test concentration of 1.0 mg mL^?1 for a 3 μL injection volume. The bulk active pharmaceutical ingredient was subjected to stress conditions of hydrolysis, oxidation, photolysis and thermal degradation. Considerable degradation was found to occur in oxidative stress conditions. The stress samples were assayed against a qualified reference standard and the mass balance was found close to 99.5%. The developed RR-LC method was validated with respect to linearity, accuracy, precision and robustness.     

Wheat digalactosyldiacylglycerol molecular species profiling using porous graphitic carbon stationary phase

The potential of porous graphitic carbon stationary phase (PGC) was assessed for the separation of molecular species of digalactosyldiacylglycerol (DGDG). Detection was by an evaporative light scattering detector (ELSD). A conventional optimization strategy allowed definition of a quaternary non-aqueous mobile phase and separation of 9 wheat DGDG molecular species with isocratic elution: methanol/toluene/tetrahydrofuran/chloroform 64.3/21.5/13.7/0.5 v/v with 0.1% of triethylamine and a stoichiometric amount of formic acid. The molecular species were identified by LC/MS. The chromatographic behavior of DGDG on PGC was then compared to previous studies. The addition of a carbon double bond on the alkyl chain decreased the retention. This contribution was less important when the number of unsaturations increased in the alkyl chain. The consequence of this retention behavior with PGC was an elution order of molecular species which did not agree with the partition number as observed with C18 grafted stationary phases.     

Determination of tryptophan and kynurenine in human plasma by liquid chromatography-electrochemical detection with multi-wall carbon nanotube-modified glassy carbon electrode

A novel method was developed for the simultaneous determination of kynurenine and tryptophan by high‐performance liquid chromatography with electrochemical detection at multi‐wall carbon nanotube (MWCNT)‐modified glassy carbon electrode. The separation and detection conditions were optimized. The typical HPLC experiments were conducted by using a reversed‐phase ODS column with a mobile phase consisting of stock acetate buffer (pH 5)–methanol (4:1, v/v) using an isocratic elution at the flow rate of 1.0 mL/min. The obtained LODs for kynurenine and tryptophane were 0.5 and 0.4 µmol/L, respectively. The analytical method for human plasma samples was validated and confirmed by LC‐UV and LC‐MS. The recoveries were in the range of 84.8–110%, and the precision was lower than 5.9%. Copyright © 2010 John Wiley & Sons, Ltd.     

Quantitation of a de‐fluorinated analogue of Casopitant Mesylate by normal‐phase liquid chromatography/mass spectrometry

The introduction of Quality by Design (QbD) in Drug Development has resulted in a greater emphasis on chemical process understanding, in particular on the origin and fate of impurities. Therefore, the identification and quantitation of low level impurities in new Active Pharmaceutical Ingredients (APIs) play a crucial role in project progression and this has created a greater need for sensitive and selective analytical methodology. Consequently, scientists are constantly challenged to look for new applications of traditional analytical techniques. In this context a normal‐phase liquid chromatography/electrospray ionization mass spectrometry (LC/ESI‐MS) method was developed to determine the amount of a de‐fluorinated analogue impurity in Casopitant Mesylate, a new API under development in GlaxoSmithKline, Verona. Normal‐phase LC provided the selectivity needed between our target analyte and Casopitant, while a single quadrupole mass spectrometer was used to ensure the sensitivity needed to detect the impurity at <0.05%w/w. Standard solutions and samples were prepared in heptane/ethanol (50:50, v/v) containing 1% of 2 M NH₃ in ethanol; the mobile phase consisted of heptane/ethanol (95:5, v/v) with isocratic elution (flow rate: 1.0 mL/min, total run time: 23 min). To allow the formation of ions in solutions under normal‐phase (apolar) conditions, a post‐column infusion of a solution of 0.1% v/v of formic acid in methanol was applied (flow rate: 200 µL/min). The analysis was carried out in positive ion mode, monitoring the impurity by single ion monitoring (SIM). The method was fully validated and its applicability was demonstrated by the analysis of real‐life samples. This work is an example of the need for selective and accurate methodology during the development of a new chemical entity in order to develop an appropriate control strategy for impurities to ultimately ensure patient safety. Copyright © 2010 John Wiley & Sons, Ltd.     

Analysis of anthranilic acid by liquid chromatography

Analytical methods for the assay of anthranilic acid and for determination of the impurities methyl anthranilate, anthranoylanthranilic acid and 3- and 4-aminobenzoic acid are described. A Microbondapak C18 column is used for both the assay and the impurity determination. The assay is based on isocratic development with a mobile phase of 35:65 v v methanol/pH-3 phosphate buffer, with benzoic acid as internal standard. The impurities are separated by gradient elution. The standard deviation of the assay method is about 1% and the limit of detection for the impurities is about 0.01%.     

Enantioseparation of tryptophan and its unnatural derivatives by nano‐LC on CSP‐teicoplanin silica based

This work deals with the potentiality of nano liquid chromatography (Nano‐LC) for the chiral separation of racemic mixture of tryptophan and some selected derivatives by using 100 µm i.d. fused silica capillary packed with teicoplanin bonded to 5 µm diol silica stationary phase. The experiments were carried out by using a cheap and laboratory‐assembled nano‐LC–UV system. Elution was done in an isocratic mode using a polar organic mobile phase. In order to find the optimum chiral separation of the studied enantiomers, some chromatographic experimental parameters were systematically studied and optimized. Among them, mobile phase composition, namely organic modifier type and concentration, buffer type and pH and aqueous content and sample solvent dilution on retention time, retention factor and enantioresolution factor were studied. Baseline enantioresolution and good peak shape was achieved utilizing the mobile phase containing 40 mM ammonium formate at pH pH 2.5 in ACN/water/acetone (60:30:10, v/v/v) at 520 nL/min in less than 8 min analysis time.     

A Validated Stability Indicating RPLC Method for Tazarotene

A simple, isocratic, rapid and accurate reversed phase high performance liquid chromatography method was developed for the quantitative determination of tazarotene. The developed method is also applicable for the related substance determination in bulk drugs. The chromatographic separation was achieved on a Hypersil C18 (250 mm × 4.6 mm 5 μm) column using water pH 2.5 with orthophosphoric acid:acetonitrile (15:85, v/v) as a mobile phase. The chromatographic resolutions between tazarotene and its potential impurity A and B were found greater than three. The limit of detection and limit of quantification of impurities were found to be 25 and 75 ng mL⁻¹. The percentage recovery of impurities in bulk drug sample was ranged from 96.8 to 103.5.The percentage recovery of tazarotene in bulk drug sample was ranged from 98.4 to 100.9. The developed RPLC method was validated with respect to linearity, accuracy, precision and robustness.