Continuous screening of analytical parameters facilitates efficient development of HPLC methods required for impurity profiling

Developing a robust analytical HPLC–UV method to characterize a drug candidate during an early stage of development is a major challenge when not all impurity standards are available. Here, we report our efforts to devise an efficient strategy for HPLC method development using continuous screening of analytical parameters without impurity standards. This strategy uses small incremental changes in the mobile phase pH and column temperature to trace each impurity on an overlay chromatogram. We tested this method using benzocaine as the active pharmaceutical ingredient (API), and compounds with similar structures to represent unknown impurities. Despite the coelution of peaks, results identified the number of impurities and indicated the starting point and parameter variables of the ensuing optimization step. Further, we demonstrated that the retention time of each peak as a function of mobile phase pH accounts for the apparent pK_a of known and unknown compounds in the presence of an organic solvent. This information is critically important to the selection of a robust pH range for HPLC methods.     

HPLC methods for the determination of simvastatin and atorvastatin

We review high-performance liquid chromatography (HPLC) methods for the determination of two major statins used in clinical treatment – simvastatin and atorvastatin – in various fields of application, including bio-analytical assays, pharmaceutical assays and environmental applications.

Statin molecules are known to be susceptible to interconversion of the lactone and acidic forms, so it is necessary to consider this phenomenon during method development. We highlight liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) methods, as they have become a method of choice in bio-analytical and environmental applications. We compare the methods from the point of view of sensitivity. We discuss selection of the precursor ion for performing selected reaction monitoring (SRM) in MS detection and sample preparation.     

Expert systems for method development and validation in HPLC

ESCA, Expert Systems Applied to Chemical Analysis, started its research in March 1987, with the aim of building prototype expert systems for HPLC method development. Results of this research have been published as the work has progressed. The project is now completed and this paper summarises some of the overall project conclusions. Seven different expert systems have been built which tackle problems throughout the process of method development, four stand-alone systems and three integrated systems. The object of ESCA was to evaluate the applicability of expert system technology to analytical chemistry and not all the systems were built for commercial uses. Many of the systems tackle problems specific to one or more of the partners and thus may not be useful outside this environment. However, the results of the work are still pertinent to analysts wishing to build their own systems. These results are described, however, the emphasis of the paper is on those systems developed for method validation.Method validation for HPLC is a complex task which requires many characteristics of the method to be tested, e.g. accuracy, precision, etc. The expert systems built within ESCA concern the validation of precision. Two systems were developed for repeatability testing and ruggedness testing. The method validation process can be divided into several discrete stages, these include: (1) The selection of the method feature to test, for instance which factors can influence the ruggedness of a method. (2) The definition of a test procedure, for instance an efficient statistical design. (3) The execution of experiments and the interpretation of results. (4) A diagnosis of any observed problem. This paper describes these two systems in some detail and summarises some of the results obtained from their evaluation. It concludes that expert systems can be useful in solving analytical problems and the integration of several expert systems can provide extremely powerful tools for the analyst.     

HPLC of nucleotides. II. General methods and their development for analysis and preparative separation. An approach to selectivity control

In order to develop efficient separation methods for nucleotides according to their size and heterocyclic composition, the application of ion-exchange, reverse-phase, and normal-phase adsorption HPLC has been studied. The comparative investigation of retention power and selectivity of various packings (non-polar bonded-phase and amino silicas) in relation to nucleotide length and composition yields data which enable suitable packings to be selected and a method of preparing the new packing for a particular separation problem to be formulated. Thus a new anion exchanger with high selectivity and dynamic mass transfer has been prepared for fractionation of large oligonucleotides. The effect of the eluent pH and composition (organic modifier, salt) on retention, selectivity, and resolution in ion-exchange and reverse-phase HPLC has been studied. The optimum separation conditions comprise elution with oppositely directed gradients of the salt and the modifier, use of a precolumn packing that provides the best protection for the main column without loss of its efficiency, and the optimum gradient program for the desired retention of the component of interest. The relation between loading and sample concentration has been studied and the system for gradient elution improved. Our work shows that two-dimensional separation is the most reliable and informative method for preparation of homogeneous oligonucleotides. The hydrophobic-pair ion-exchange mechanism is proposed for ion-pair chromatography. Protected and partially deblocked oligonucleotides, chemically synthesized for genetic engineering studies, have been separated with high selectivity by adsorption (normal-phase) HPLC which is efficient for gradient elution with isohydric eluents. The analysis of a monomeric composition of nano-(pico-) molar amounts of oligonucleotides has been developed; the procedure involves microcolumn digestion of the oligonucleotides with immobilized enzymes followed by microcolumn separation of the nucleoside-mononucleotide mixture. Also, a new slurry method for packing stable HPLC columns with a tightly consolidated, nonshrinkable bed of particles has been developed.     

Review of HPLC methods and HPLC methods with mass spectrometric detection for direct determination of aspirin with its metabolite(s) in various biological matrices

Aspirin, the most widely used drug in the world, has been known to mankind for over a century. It is not only the pharmacologically active entity, but is also biotransformed into a major metabolite, i.e. salicylic acid, which also exhibits similar pharmacologic/pharmacodynamic properties. Hence it is necessary to quantitate aspirin along with its metabolite(s) in various biological matrices accurately and precisely to correlate with pharmacological/pharmacodynamic activity. This paper provides a comprehensive overview of various bioanalytical methods (HPLC and LC-MS/MS) that have been reported for direct quantitation of aspirin along with its metabolite(s). The review also provides general information on sample collection, sample processing, internal standard selection, conditions for chromatographic separation, succinct validation data and applicable conclusions for reported assays in a structured manner.     

Fingerprint analysis of Shankhpushpi for species discrimination by HPLC coupled with chemometric methods

Abstract

Shankhpushpi, traded in the Indian market is having different controversial botanical sources, that is, Convolvulus pluricaulis Choisy (Convulvulaceae), Clitoria ternatea Linn. (Papilionaceae), and Evolvulus alsinoides Linn. (Convulvulaceae). Simple, accurate and precise RP-HPLC-PDA method was established for quality assessment and discrimination of Shankhpushpi samples using chromatographic profiling method. In distinction to the routine method of quality assessment which uses single or dual marker peaks, all chromatographic data (retention times/variables) were used. Fifteen shankhpushpi samples were purchased from the market including authenticated samples of all three C. pluricaulis, C. ternatea, E. alsinoides. A total of 18 samples were analyzed by HPLC and the dataset was then treated with multivariate analyses like PCA and HCA by using MINITAB software. Thus, the developed method was useful in discriminating the Shankhpushpi samples and for the perseverance of quality control.     

Requirements and tests for HPLC apparatus and methods in pharmaceutical quality control

Summary One possible method has been demonstrated for pharmaceutical quality control which is not restricted to this field and fulfils and documents the prerequisites for reliable, accurate and precise HPLC analysis. This includes validation which shows that the method is able in principle to fulfil the requirements, the apparatus test which shows that the apparatus generally works correctly and precisely and the system suitability test which shows that the method provides accurate and precise results on this apparatus and with this column for the analysis in question when other non-equipment and non-method-induced errors are excluded.     

Comparison of different extraction and detection methods for sugars using amino-bonded phase HPLC

In this study, different methods are compared in order to quantitate individual sugars extracted from apple samples using methanol and water. Glucose, fructose, and sucrose are separated in 20 min using an amino-bonded carbohydrate column and a 75:25 acetonitrile-water mobile phase, followed by UV (190 nm) and refractive index detection. Variations in the sugar profiles are observed using different extraction or detection methods (or both) at a 1.4-mL/min flow rate. The data obtained show differences (p < 0.01) from both extraction or detection methods. The highest contents of free sugars studied occur in samples extracted with water.     

Spectroscopic and HPLC methods for the determination of alendronate in tablets and urine

Two methods (spectrophotometric and HPLC) have been developed and validated for the analysis of alendronate sodium in tablet dosage form. Both methods depend on the ability of alendronate sodium to react with o-phthalaldehyde (OPA) at basic pH to produce a light-absorbing derivative. The derivative was found to possess absorption maximum at 330 nm where neither the derivatizing agent nor the analyte had any absorption. Thus, spectroscopic method was based on the derivatization-induced absorption of alendronate sodium at 333 nm. The HPLC method was based on separation of the formed derivative from other ingredients in tablets with detection at 333 nm. Both methods were satisfactory with regard to accuracy, prescion and linearity. Moreover, a HPLC method with fluorescence detection (HPLC-FD) was developed for the quantification of alendronate sodium in urine. The method was also based on the derivatization of alendronate with OPA, but fluorescence detection was employed. Linearity, recovery, selectivity, prescision and sensitivity were satisfactory for the proposed HPLC-FD method. Yet a new quantification limit (0.6 ng ml⁻¹) for alendronate in urine was achieved.     

Gold nanoparticles for the development of clinical diagnosis methods

The impact of advances in nanotechnology is particularly relevant in biodiagnostics, where nanoparticle-based assays have been developed for specific detection of bioanalytes of clinical interest. Gold nanoparticles show easily tuned physical properties, including unique optical properties, robustness, and high surface areas, making them ideal candidates for developing biomarker platforms. Modulation of these physicochemical properties can be easily achieved by adequate synthetic strategies and give gold nanoparticles advantages over conventional detection methods currently used in clinical diagnostics. The surface of gold nanoparticles can be tailored by ligand functionalization to selectively bind biomarkers. Thiol-linking of DNA and chemical functionalization of gold nanoparticles for specific protein/antibody binding are the most common approaches. Simple and inexpensive methods based on these bio-nanoprobes were initially applied for detection of specific DNA sequences and are presently being expanded to clinical diagnosis. Figure Colorimetric DNA/RNA detection using salt induced aggregation of AuNP-DNA nanoprobes.     

Development and validation of stability-indicating HPLC and UPLC methods for the determination of bicalutamide

Six new process related impurities (Imp-08, Imp-09, Imp-10, Imp-12, Imp-13 and Imp-14) of bicalutamide (BCT) have been reported in this paper. BCT was subjected to oxidative, acid, alkaline, hydrolytic, thermal and photolytic degradation conditions and found to degrade in alkaline condition, yielding Imp-11. Stability-indicating high-performance liquid chromatography and ultra-performance liquid chromatography methods were developed for the determination of BCT in the presence of its 14 process-related impurities and 1 degradant by using Zorbax SB phenyl column (150 × 4.6 mm × 3.5 μm) and HSS T3 column (100 × 2.1 mm × 1.8 μm), respectively. Both the methods were validated as per International Conference on Harmonization guidelines. Quantitation limits (QL) were found be in the ranges of 0.02-0.03% for both the methods. Precision was evaluated by replicate analysis in which % relative standard deviation (RSD) values for areas were found below 2.0. Linearity for the impurities was established in the range of QL to 200% of the specification level and the correlation coefficients derived from of the respective calibration curves were approximately 0.999. The recoveries obtained for purity (90-100%) and assay (98-102%) ensured the accuracy of the developed methods.     

Historical development of thermoanalytical methods

Thermal tests in antiquity and in the Middle Ages. Blowpipe tests. Measuring temperature and the development of thermometers. Latent heat. The nature of heat. Amount of heat. Differential thermal analysis. Differential scanning calorimetry. Heat treatment of precipitates. Thermobalances. Thermoanalytical methods associated with determination of evolved gas. Thermometry, enthalpimetry.     

Analytical QbD-based systematic bioanalytical HPLC method development for estimation of quercetin dihydrate

The current work entails development of rapid, sensitive, and inexpensive high-performance liquid chromatographic method of quercetin dihydrate using the quality by design approach. Quality target method profile was defined and critical analytical attributes (CAAs) were earmarked. Chromatographic separation was accomplished on a C₁₈ column using acetonitrile and ammonium acetate buffer (35:65) %v/v (containing 0.1% acetic acid, pH 3.5) as mobile phase at 0.7?mL/min flow rate with UV detector at 237?nm. Screening studies using fractional factorial design revealed that organic modifier, injection volume, column temperature, and buffer strength have significant influence on method CAAs, namely, peak area, retention time, and peak tailing. The critical method parameters were systematically optimized using Box–Behnken design. Response surface mapping was used along with numerical optimization and desirability function for identifying the optimal chromatographic conditions. Linearity was observed in the drug concentration ranging between 2 and 50?µg/mL. Accuracy analysis revealed mean % recovery between 93.6 and 96.2%, while precision study revealed mean % recovery between 93.7 and 96.5%. Limits of detection and quantification of the developed method were found to be 12.1 and 36.6?ng/mL. Overall, the studies construed successful development of chromatographic method of quercetin with enhanced method performance.     

Microplate-based screening methods for the efficient development of sandwich immunoassays

The selection of suitable antibodies is a critical step in immunoassay development, since the final assay performance is predetermined by this decision to a large extent. Particularly, the screening for matching pairs in sandwich immunoassays is difficult, if both antibodies are derived from one species or when monoclonal antibodies are only available as cell supernatants. Several microplate-based approaches for in situ labeling of detection antibodies were tested, in order to avoid time consuming purification of antibodies for enzyme conjugate synthesis. We investigated labeling with anti-species antibodies and Fab fragments thereof, labeling with protein G and biotinylation of cell supernatants without prior purification. Antibodies against peanut proteins were used as a model and signal-to-blank ratios were used in all cases as a measure of the antibody pair performance. Amongst the investigated approaches, preincubation of the detection antibody with labeled anti-species antibody turned out to be most suitable under our conditions. Diagrams, showing the performance of all possible antibody combinations, were generated with this method and were compared to results obtained with covalently labeled detection antibodies. Finally, a flowchart is presented, suggesting an efficient strategy for the development of highly sensitive sandwich immunoassays.     

Characterization of nutraceuticals and functional foods by innovative HPLC methods

In recent years there is a growing interest in food and food ingredient which may provide health benefits. Food as well as food ingredients containing health-preserving components, are not considered conventional food, but can be defined as functional food. To characterise such foods, as well as nutraceuticals specific, high sensitive and reproducible analytical methodologies are needed. In light of this importance we set out to develop innovative HPLC methods employing reversed phase narrow bore column and high-performance anion-exchange chromatographic methods coupled with pulsed amperometric detection (HPAEC-PAD), which are specific for carbohydrate analysis. The developed methods were applied for the separation and quantification of citrus flavonoids and to characterize fructooligosaccharide (FOS) and fructans added to functional foods and nutraceuticals.     

Comparison of HPLC and multivariate regression methods for hydrocortisone and lidocaine analysis of pharmaceutical preparations

A reverse-phase high-performance liquid chromatographic (HPLC) method to determine hydrocortisone acetate, hydrocortisone hemisuccinate and lidocaine is described in this paper. The separation was made in a LichrCART C(18) column using a methanol-NaH(2)PO(4)/Na(2)HPO(4) (0.1 mol L(-1)) (pH=4.5) buffer solution as a mobile phase in isocratic mode (60:40 (v/v)). The mobile phase flow rate and the sample volume injected were 1 mL min(-1) and 20 micro L, respectively. The detection was made with a diode-array detector measuring at the maximum for each compound. Quantification limits ranging from 0.18 to 0.84 micro g L(-1) were obtained when the peak area was measured. The method was applied in pharmaceutical formulations that were compared with those obtained by through multivariate regression spectrophotometry and micellar capillary electrophoresis (MEKC). HPLC results are in accordance with the results obtained by MEKC. The spectrophotometric method was suitable only for synthetic samples.