Alexander H. Schmidt

Computer-assisted optimization in the development of a high-performance liquid chromatographic method for the analysis of kava pyrones in Piper methysticum preparations.

A computer simulation program was used to optimize the separation for six kava pyrones and two unidentified components obtaining the best resolution and the shortest run time. With DryLab it was possible to find the best separation conditions without running a large number of possible combination of variables in the laboratory. Additionally, due to the systematic progress in method development a new eluent was found with excellent properties, namely 2-propanol. With 2-propanol, the largest number of components could be revealed in the shortest analysis time. Starting with four initial experiments, the software allowed to optimize gradient time tG and temperature T simultaneously. Changing other variables such as type of organic modifier, the eluent pH, the gradient form, and the flow-rate, the optimization resulted in resolution Rs > 1.5 for all kava pyrones and the two additional new bands. The HPLC method is used to analyze kava pyrones in Piper methysticum preparations.

Validated HPLC Method for the Determination of Residues of Acetaminophen, Caffeine, and Codeine Phosphate on Swabs Collected from Pharmaceutical Manufacturing Equipment in Support of Cleaning Validation †

Abstract A high performance liquid chromatography (HPLC) method was developed for the simultaneous determination of residues of acetaminophen (paracetamol), caffeine, and codeine phosphate on swabs collected from pharmaceutical manufacturing equipment surfaces. Any residues of the compounds remaining on process equipment after cleaning are removed by swabbing with wet Texwipe® swabs, premoistened with methanol/water, followed by dry Texwipe® swabs. These residues are extracted from the swabs by means of an ultrasonic bath and the amounts of the compounds are determined. The chromatography was performed in the isocratic mode on a RP‐18 column using a mobile phase consisting of 25 mM ortho‐phosphoric acid and acetonitrile (90:10, v/v). UV‐ and fluorescence detection was performed in order to improve the methods sensitivity. The method was validated by specificity, linearity, limit of detection, and limit of quantification, accuracy, and precision for the residues of acetaminophen (paracetamol), caffeine, and codeine phosphate on equipment surfaces. Stability studies have demonstrated the stability of the residual active compounds on equipment surfaces and on the swabs. †Parts of this paper were presented at the 29th International Symposium on High Performance Liquid Phase Separations & Related Techniques, “HPLC 2005”, June 26–30, 2005, Stockholm, Sweden.

SFC-MS/MS as an orthogonal technique for improved screening of polar analytes in anti-doping control

AbstractHPLC is considered the method of choice for the separation of various classes of drugs. However, some analytes are still challenging as HPLC shows limited resolution capabilities for highly polar analytes as they interact insufficiently on conventional reversed-phase (RP) columns. Especially in combination with mass spectrometric detection, limitations apply for alterations of stationary phases. Some highly polar sympathomimetic drugs and their metabolites showed almost no retention on different RP columns. Their retention remains poor even on phenylhexyl phases that show different selectivity due to π–π interactions. Supercritical fluid chromatography (SFC) as an orthogonal separation technique to HPLC may help to overcome these issues. Selected polar drugs and metabolites were analyzed utilizing SFC separation. All compounds showed sharp peaks and good retention even for the very polar analytes, such as sulfoconjugates. Retention times and elution orders in SFC are different to both RP and HILIC separations as a result of the orthogonality. Short cycle times could be realized. As temperature and pressure strongly influence the polarity of supercritical fluids, precise regulation of temperature and backpressure is required for the stability of the retention times. As CO2 is the main constituent of the mobile phase in SFC, solvent consumption and solvent waste are considerably reduced. Graphical AbstractSFC-MS/MS vs. LC-MS/MS

Life cycle management of analytical methods

Graphical abstract Figure. No Caption available. HighlightsLife cycle management as novel strategy for analytical methods.Quality‐by‐design principles for analytical methods.Integrated method development and performance characterization approach.Proposal of new guidelines for regulated environment in progress. ABSTRACT In modern process management, the life cycle concept gains more and more importance. It focusses on the total costs of the process from invest to operation and finally retirement. Also for analytical procedures an increasing interest for this concept exists in the recent years. The life cycle of an analytical method consists of design, development, validation (including instrumental qualification, continuous method performance verification and method transfer) and finally retirement of the method. It appears, that also regulatory bodies have increased their awareness on life cycle management for analytical methods. Thus, the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH), as well as the United States Pharmacopeial Forum discuss the enrollment of new guidelines that include life cycle management of analytical methods. The US Pharmacopeia (USP) Validation and Verification expert panel already proposed a new General Chapter ⟨1220⟩ “The Analytical Procedure Lifecycle” for integration into USP. Furthermore, also in the non‐regulated environment a growing interest on life cycle management is seen. Quality‐by‐design based method development results in increased method robustness. Thereby a decreased effort is needed for method performance verification, and post‐approval changes as well as minimized risk of method related out‐of‐specification results. This strongly contributes to reduced costs of the method during its life cycle.

A QBD WITH DESIGN-OF-EXPERIMENTS APPROACH FOR DEVELOPMENT OF A STATE-OF-THE-ART UPLC PURITY METHOD FOR CARBAMAZEPINE

A state-of-the-art ultra-performance liquid chromatographic (UPLC) method has been developed for purity testing of carbamazepine. Successful chromatographic separation of the active pharmaceutical ingredient (API) from its impurities was achieved on a WATERS ACQUITY UPLC CSH C18 column with the dimensions 2.1 mm × 100 mm and 1.7 µm particle size with gradient elution of 0.2% phosphoric acid and acetonitrile in only 5 min. Incorporating Quality by Design (QbD) principles to the method development approach by using the statistical software package Fusion AE allows the study of the relationship between chromatographic parameters (factors) and the resolution (response) between the peaks of interest. In a screening phase, the factors known to have a major effect on column selectivity (stationary phase, pH of the aqueous eluent, organic eluent type, gradient time, and slope) were studied. In the second phase, the chromatographic parameters that were identified as affecting the resolution were studied with additional instrument settings. In both phases, statistical concepts with experimental design plans (Design-of-Experiments) are used as an efficient and fast tool to simultaneously gain knowledge regarding the influencing factors and interactions. An operating space within the design space was established and a verification study confirmed the robustness of the final method. Total analysis time was only 5 min, which is an impressive 22-fold increase in productivity in comparison to the method published in the European Pharmacopeia.

Development of an HPLC Method for the Determination of Hydroxycinnamic Acid Derivatives in Cimicifuga racemosa (Black Cohosh) Extracts Using an Automated Method Development System

Abstract The separation of a complex mixture, such as the ingredients in medicinal plants, is typically difficult and the development of a HPLC method is a labor‐intensive and time‐consuming process if carried out manually. Automation of this process can increase productivity of a pharmaceutical R&D department substantially. This paper describes the development of a high performance liquid chromatographic method for the determination of hydroxycinnamic acid derivatives in Cimicifuga racemosa extracts and its preparations by using a fully automated method development system (Waters AMDS). The developed method is based on the baseline chromatographic separation of six hydroxycinnamic acid derivatives (caffeic acid, ferulic acid, isoferulic acid, fukinolic acid, cimicifuga acid A, and cimicifuga acid B), the major constituents in Cimicifuga racemosa (Black Cohosh), on a XTerra MS C18 column with a water‐methanol gradient and photodiode array detection.

Distinction of clenbuterol intake from drug or contaminated food of animal origin in a controlled administration trial – the potential of enantiomeric separation for doping control analysis

ABSTRACT The differentiation of clenbuterol abuse and unintentional ingestion from contaminated meat is crucial with respect to the valuation of an adverse analytical finding in human sports doping control. The proportion of the two enantiomers of clenbuterol may serve as potential discriminating parameter. For the determination of the individual enantiomers, specific methods were developed and validated for the different matrices under investigation based on chiral chromatography coupled to tandem mass spectrometry. Data are presented from the administration to humans of clenbuterol from a pharmaceutical preparation, and from cattle meat and liver containing residues. A shift in the proportion of the enantiomers in cattle meat is detected and this signature is also found in human urine after ingestion. Thus, an altered enantiomeric composition of clenbuterol may be used to substantiate athletes’ claims following adverse analytical findings in doping control. However, in meat, the enantiomeric composition was found to be highly variable. Species as well as tissue dependent variances need to be considered in interpreting enantiomer discrimination. Analysis of post administration urines from a controlled experiment comparing the administration of racemic clenbuterol from a registered pharmaceutical preparation and the administration of residue-containing meat and liver (nonracemic mixture) from treated animals is reported. Furthermore doping control samples from Mexican U17 World Championship 2011 of the Fédération Internationale de Football Association (FIFA), with adverse analytical findings for clenbuterol, were re-analysed. GRAPHICAL ABSTRACT

What is the potential of measuring the enantiomeric ratio of drugs using supercritical fluid chromatography–MS?

Analytical chemists are always looking for more efficient techniques to meet analytical challenges of today’s regulatory and scientific requirements. One technique that has made drastic improvements in recent years is supercritical fluid chromatography (SFC). Since its early days, this separation technique has emerged as a powerful tool for both analytical and preparative scientists. In 2012, new instruments for analytical purpose were introduced that offer reliability, robustness and sensitivity never before possible. Since then, the interest in this technique was renewed. Indicated by the number of peerreviewed articles, analytical SFC progressively increased within the last 25 years. The use of supercritical fluids (sc) as mobile phases in chromatography presents unique advantages compared with traditional HPLC: low viscosity and high diffusivity allow faster and more efficient separations than HPLC. Coupling SFC to MS instruments is straightforward as carbon dioxide is easily eliminated during desolvation and provides low LODs and LOQs as well as additional structural information [1]. A wide range of analytes may be covered by SFC, including nonpolar, polar and charged compounds [2]. Applications for a broad variety of polarity are reported ranging from hydrocarbons, esters, amines, aldehydes, alcohols, carboxylic acids, amides, amphoterics, complexes to peptides. In general, supercritical carbon dioxide (CO 2,sc ) is used as mobile phase due to its easy generation and low toxicity. The more polar analytes can be separated by addition of modifiers and additives (mainly methanol and also even water). Thus, SFC is emerging as a technique of choice for enantioselective separations and as replacement for HPLC methods [3]. A positive side effect is the reduction of toxic solvents compared with HPLC, which can minimize waste and decrease the cost of chiral separations significantly.

Validation of A Fast‐HPLC Method for the Separation of Iridoid Glycosides to Distinguish Between the Harpagophytum Species

Abstract A fast high‐performance liquid chromatography (HPLC) method was developed and validated for the simultaneous determination of the iridoid glycosides harpagoside (HS) and 8‐p‐coumaroyl‐harpagide (8pCHG) in extracts and preparations of Harpagophytum procumbens and H. zeyheri. The ratio between 8pCHG and the sum of HS and 8pCHG can be used to distinguish between both species. Quantitation was accomplished with the internal standard (IS) method. The separation was performed on a monolithic silica column (Chromolith Performance RP‐18e), under gradient conditions using a mobile phase of water (pH 2.0 adjusted with phosphoric acid) and acetonitrile. The elution of the analytes was monitored at 278 nm and conducted at a column temperature of 30°C. Because of the high porosity of the monolithic column the mobile phase was able to be pumped at a flow rate of 5.0 mL/min. The retention time of 8pCHG, HS, and the IS was 1.9 min, 2.1 min, and 3.0 min, respectively, and the total run time of the assay was 5 min. The method was validated by specificity, linearity, accuracy, and precision. For the determination of method robustness a number of chromatographic parameters were varied.

Structure assisted impurity profiling for rapid method development in liquid chromatography

The use of trial-and-error principles is a frequently used technique in method development. This may lead to the fact that analytical methods are used routinely without developers and users having gained extensive and well-founded knowledge about the robustness of their analytical methods and the influence of critical key parameters. This very often leads to unnecessary problems for analysts. A simple way in reverse phase chromatography to simulate the effects of pH value changes on the separation and retention of substances is the pH-dependent calculation of the logD value. With this tool, model substances were used to show how the time requirement for method screening can be considerably reduced in silico and, in addition, extended knowledge about the separation mechanics can be generated. Based on this knowledge, a new method for the purity analysis of carbamazepine was developed within a very short period of time, which improves the performance of the official Ph.Eur. monograph by far. Furthermore, the extremely high robustness of the new method was demonstrated. Using the logD based approach, Quality-by-Design is applied in method development and kept pace with the increasing requirements of regulatory authorities in the pharmaceutical industry.