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Retention modeling of therapeutic peptides in sub-/supercritical fluid chromatography

Jonas Neumann, Sebastian Schmidtsdorff, Alexander H. Schmidt, Maria K. Parr

Separation Science Plus published by Wiley-VCH GmbH 11 February 2024

Chromatographic modeling software packages are valuable tools during method optimization steps. These are well established for reversed-phase applications utilizing retention time (RT) prediction to optimize separations and receive robust methods, which is of high interest for the analysis of pharmaceuticals. In contrast to liquid chromatography, the knowledge of RT prediction in supercritical fluid chromatography is limited to a manageable number of studies.

This study uses the software DryLab to predict the RTs of the peptides bacitracin (Bac), colistin, tyrothricin (Tyro), and insulin analogs. Gradient time, column temperature, and the ternary composition (terC) of carbon dioxide, methanol (MeOH), and acetonitrile (ACN) in the gradient elution are varied in a feasibility approach using a neutral (Viridis BEH) and an amino-derivatized aromatic (Torus 2-PIC) stationary phase. In the second part, chromatographic optimization is performed in silico through gradient adjustments to optimize the separation of the fingerprint of Bac. The final gradient method utilizes a Viridis BEH column (100 × 3.0 mm, 1.7 μm), carbon dioxide, and a modifier consisting of ACN/MeOH/water/methanesulfonic acid (60:40:2:0.1, v:v:v:v). In addition, changes in the retention order of Tyro compounds with the proportion of the terC in combination with a Torus Diol column are investigated.

ICH Q14-inspired novel approach to establish an SFC-based purity method for carbamazepine

Alexander H. Schmidt, Mijo Stanic, Maria Kristina Parr

Drug Testing and Analysis published by John Wiley & Sons Ltd. 15 January 2024

The proposed ICH Q14 guideline “Analytical procedure development” describes science and risk-based approaches for development and maintenance of analytical procedures suitable for the assessment of the quality of drug substances and drug products. As a case study, the systematic development and validation of a supercritical fluid chromatography (SFC)-based purity method for carbamazepine is presented. Systematic analytical quality by design (AQbD) principles were applied using the software package Fusion QbD to the method development approach. The relationship between chromatographic parameters and the responses of interest were examined to improve the reliability of the method by understanding, reducing, and controlling sources of variability. Method performance qualification in terms of method robustness was finally carried out with the parameters that were classified as critical after method development and a validation study met previously set acceptance criteria. The developed SFC purity method for carbamazepine demonstrated readiness as a viable alternative to the official HPLC method published in the Ph.Eur. with improved peak resolution, improved peak symmetry, and faster analysis times (3 min vs. 80 min for the official method). Its inherent reliability illustrates the superiority of AQbD in method development and application for drug quality assurance.

Controlling the elution order of insulin and its analogs in sub-/supercritical fluid chromatography using methanesulfonic acid and 18-crown-6 as mobile phase additives

Jonas Neumann, Sebastian Schmidtsdorff, Alexander H. Schmidt, Maria K. Parr

Journal of Separation Science Volume 46 Issue 21 29. Sep. 2023

The purity analysis of therapeutic peptides can often be challenging, demanding the application of more than a single analytical technique. Supercritical fluid chromatography nowadays is a promising alternative to reversed-phase liquid chromatography, providing orthogonal and complementary information. This study investigated its applicability for the separation of human insulin, its analogs and degradation products. A previously published method development protocol for peptides up to 2000 Da was successfully applied to the higher molecular weight insulins (6 kDa). A single gradient method was optimized for all insulins using a Torus DEA column (100 × 3.0 mm, 1.7 μm), carbon dioxide and a modifier consisting of methanol/acetonitrile/water/methanesulfonic acid (65:35:2:0.1, v/v/v/v). Consecutively, the crown ether 18-crown-6, which is well known to complex charged lysine sidechains and other amino functionalities, was added to the modifier to evaluate its impact on selectivity. A decreased retention and a shift in the elution order for the insulins were observed. An inverse effect on retention was found when combined with a neutral stationary phase chemistry (Viridis BEH).

Ternary eluent compositions in supercritical fluid chromatography improved fingerprinting of therapeutic peptides

Jonas Neumann, Sebastian Schmidtsdorff, Alexander H. Schmidt, Maria K. Parr

Journal of Separation Science 05 January 2023

Currently, little information has been published on the application of ternary eluent compositions in supercritical fluid chromatography for separating peptides. This work investigates the benefits of adding acetonitrile to methanol as the modifier. Three cyclic antibiotic peptides (bacitracin, colistin, and daptomycin) ranging between 1000 and 2000 Da were chosen as model substances. The ternary mixture of carbon dioxide, methanol, and acetonitrile is optimized to increase the resolution of the peptide’s fingerprint. In addition, varying compositions of methanol and acetonitrile were found to change the elution order of the analytes, which is a valuable tool during method development. An individual gradient method using two Torus 2-PIC columns (each 100 × 3.0 mm, 1.7 μm), carbon dioxide, and a modifier consisting of acetonitrile/methanol/water/methanesulfonic acid (60:40:2:0.1, v:v:v:v) was optimized for each of the peptides. Subsequently, a generic method development protocol applicable to polypeptides is proposed.

Prevalence of nitrosamine contaminants in drug samples: Has the crisis been overcome?

Sebastian Schmidtsdorff, Jonas Neumann, Alexander H. Schmidt, Maria K. Parr

Arch. Pharm,. 02 December 2022

Various drug samples (N = 249; drug substances, tablets, capsules, solutions, crèmes, and more) from the European pharmaceutical market were collected since 2019 and analyzed for 16 nitrosamines (NAs). In 2.0% of the cases, NAs were detected. These findings included four active pharmaceutical ingredients already known for potential NA contamination: losartan (N-nitrosodimethylamine [NDMA] and N-nitrosodiethylamine, simultaneously), valsartan (NDMA), metformin (NDMA) and ranitidine (NDMA). The fifth new finding, which has not been reported yet, discovered contamination of a molsidomine tablet sample with N-nitrosomorpholine (NMor). The tablet contained 144% of the toxicological allowable intake for NMor. NMor was included in our screening from the beginning and is currently the focus of regulatory authorities, but was added to the guidelines only last year. Thus, it may not have been the focus of regulatory investigations for too long. Our results indicate that the majority of drug products in the market are nonhazardous in terms of patient safety and drug purity. Unfortunately, the list of individual affected products keeps growing constantly and new NA cases, such as molsidomine or nitrosated drug substances (nitrosamine drug substance-related impurities [NDSRI]), continue to emerge. We therefore expect nitrosamine screenings to remain a high priority.

Application of sub-/supercritical fluid chromatography for the fingerprinting of a complex therapeutic peptide

Jonas Neumann, Sebastian Schmidtsdorff, Alexander H. Schmidt, Maria K. Parr

Arch. Pharm,. 06 July 2022 –

The application area of supercritical fluid chromatography expanded tremendously over the last years and more polar analytes such as biomolecules have become accessible. The growing interest in biopharmaceuticals and associated regulatory requirements demand alternative analytical tools. The orthogonal nature of supercritical fluid chromatography compared to reversed-phase liquid chromatography meets these needs and makes it a useful option during research and development.

In this study, we present a systematic approach for the development of a supercritical fluid chromatography method for fingerprinting of tyrothricin, a complex therapeutic peptide covering a mass range from 1200 to 1900 Da. The substance was chosen due to the presence of cyclic and linear peptides and isomeric or highly similar amino acid sequences. Different column chemistries covering neutral, basic, and zwitterionic functionalities in combination with acidic, basic, and neutral additives were screened. Subsequently, Design-of-Experiments principles were utilized to perform optimization of the chromatographic parameters. The final mass spectrometry-compatible gradient method using a diol stationary phase, carbon dioxide, and a modifier consisting of methanol/water/methanesulfonic acid (100:2:0.1, v:v:v) was found to provide orthogonality and superior resolution to other methods published. Isomeric peptide compounds coeluting in reversed-phase liquid chromatography were resolved by applying the final method.

Risk assessment for nitrosated pharmaceuticals: A future perspective in drug development

Sebastian Schmidtsdorff, Jonas Neumann, Alexander H. Schmidt, Maria K. Parr

Arch. Pharm. – 27 January 2022

Since June 2018, thousands of drug products from around the world had to be recalled due to the unexpected presence of nitrosamines (NAs). Starting with the pharmaceutical group of sartans, antidiabetic drugs, antihistamines, and antibiotics also became the subject of investigation. The occurrence of NAs has shown that pharmaceutical companies and regulatory agencies did not focus on these substances in the past during drug development. In this study, we incorporated a nitrosation assay procedure into high-resolution supercritical fluid chromatography (SFC)–mass spectrometry screening to test the potential of direct nitrosation of active pharmaceutical ingredients (APIs). The forced degradation study was performed with a four-fold molar excess of sodium nitrite, relative to the drug substance, at pH 3–4 for 4 h at 37°C. Chromatographic separation was performed on a porous graphitic carbon column by SFC. The mass analysis then focused on direct N-nitrosation or N-nitroso compounds (NOCs) formed after dealkylation. Substances (n = 67) from various pharmaceutical classes were evaluated and 49.3% of them formed NOCs, of which 21.2% have not yet been reported in the literature. In addition, for two APIs, which are known to form an unidentified NOC, the structure could be identified. A few substances also showed multiple NOCs and even N,N’-dinitroso-species. As NAs are carcinogens, they have to be eliminated or at least limited to prevent cancer in patients, who rely on these drugs. This study contributes a procedure that can be implemented in preapproval drug development and postapproval risk assessment to prevent unexpected findings in the future.

Analytical lifecycle management for comprehensive and universal nitrosamine analysis in various pharmaceutical formulations by supercritical fluid chromatography

Sebastian Schmidtsdorff, Jonas Neumann, Alexander H.Schmidt, Maria K.Parr

Journal of Pharmaceutical and Biomedical Analysis 197 (2021) 113960

Since the detection of nitrosamines (NA) in valsartan pharmaceuticals, over two years have passed. At present, the occurrence of NAs can be limited to a few drug substances and drug products, but it is already becoming apparent that the issue appears to be much bigger than initially thought. The impact on the global pharmaceutical market has been tremendous and the problem can be attributed mainly to uncritically adopted approval changes and the lack of suitable, modern analytical methods to detect those impurities in time.

We hereby demonstrate how lifecycle management (LCM) can be used to develop and improve suitable and universal analytical methods within short time. The resulting SFC-MS/MS method is intended for a universal nitrosamine investigation in drug substances and drug products. Successful NA analysis was demonstrated for seven sartans, metformin, pioglitazone and ranitidine. Additionally, combination drug products, containing also amlodipine, hydrochlorothiazide, vildagliptin and sitagliptin, were analyzed successfully. The method achieved separation of 16 NAs in 4 min with a total run time of 11.5 min, utilizing a Supel Carbon porous graphitic carbon (PGC) column. Carbon dioxide together with 0.1% TFA in methanol as modifier were used as eluents and 0.35% formic acid in methanol as make-up solvent for mass spectrometric NA detection. By implementing LCM in this case study, development time was reduced and knowledge was implemented fast. At the same time, a high adaptability of this “vital” method was achieved, which makes it possible to implement the constantly changing regulatory requirements within the shortest possible time. Supplemental development data, according to the ICH guidelines Q8, Q12 and the proposed Q14 are disclosed, demonstrating the scientific Quality-by-Design (QbD) development approach, the “fitness for use” and the robustness of the analytical procedure.

This method contributes to the still ongoing risk assessment process of the pharmaceutical industry and the regulatory agencies, in order to understand root causes of NA formation, maintain the drug supply and prevent drug shortage.

Simultaneous detection of nitrosamines and other sartan-related impurities in active pharmaceutical ingredients by supercritical fluid chromatography

Sebastian Schmidtsdorff, Alexander H.Schmidt

Journal of Pharmaceutical andBiomedical Analysis Volume 174, 10 September 2019, Pages 151-160

Since July 2018, the pharmacological class of “sartans” has been the subject of considerable media and analytical interest, as it became known that they are contaminated with nitrosamines such as N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA) and N-nitrosodiisopropylamine (NDiPA). Previous compendial methods are not able to detect these new contaminants. Using the latest and innovative Quality-by-Design (QbD) approach, it has now been possible to develop an analytical method that enables to investigate sartans, such as valsartan and losartan. Also a large class of different nitrosamines in the ppb range and sartan-related impurities can thus be determined simultaneously in a single analysis using supercritical fluid chromatography (SFC). By using SFC, a broad spectrum of nonpolar and very polar impurities can be separated and analyzed in under 20 min. The analytical method developed is validated for limit testing according to ICH Q2(R1) and fulfills default thresholds of EMA and FDA for testing of drug substances and genotoxic impurities. Additionally, it can also be adapted to other pharmaceuticals that may be contaminated with nitrosamines, since tetrazole synthesis as the underlying cause of nitrosamine contamination is important for a set of other non-sartan drug substances.

Robustness Modelling in Ultrahigh-Pressure Liquid Chromatography Methods

Imre Molnár, Alexander H. Schmidt

The Column, Volume 14, Issue 7, pg 21–28 (2018), 2018

Many workers in pharmaceutical laboratories are unable to change any aspect of their methods, although they often encounter severe problems and create many out-of-specification (OoS) results. They are particularly afraid to investigate these problems from a chromatographic perspective in case they generate new unforeseen problems. In the literature, however, there are numerous examples showing that it is worthwhile trying to understand the reasons for “unexplainable” behaviour in ultrahigh-pressure liquid chromatography (UHPLC) using modelling. By using modelling, problems can be recognized and often eliminated with legal operations according to the allowed tolerance limits mentioned in pharmacopoeia descriptions. The following article aims to show that “visual chromatographic modelling” can be a useful aid.

Structure assisted impurity profiling for rapid method development in liquid chromatography

Sebastian Schmidtsdorff, Alexander H. Schmidt, Maria Kristina Parr

Journal of Chromatography A, 2018

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.

Keywords: Distribution coefficient (logD, Method development, Quality-by-design (QbD), High-performance liquid chromatography (HPLC), Chromatographic modeling, Extended knowledge space

Review: Life cycle management of analytical methods

Maria Kristina Parr, Alexander H. Schmidt

Journal of Pharmaceutical and Biomedical Analysis (2017)

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-regutated environment a growing interest on life cycle management is seen. Qyality-by-design based method development results in increased method robustness. Thereby a decreased effort is needed for method performance verification, and postapproval 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.

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

Maria Kristina Parr, Bernhard Wuest, Edgar Naegele, Jan F. Joseph, Maxi Wenzel, Alexander H. Schmidt, Mijo Stanic, Xavier de la Torre, Francesco Botrè

Anal Bioanal Chem (2016) 408:6789–6797

HPLC 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.

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

Parr, Maria Kristina; Blokland, Marco; Liebetrau, Franz, Meijer, Thijs; Schmidt, Alexander; Stanic, Mijo; Kwiatkowska, Dorota; Waraksa, Emilia; Sterk, Saskia

Food Additives & Contaminants: Part A,34 (2017) 525-535 Year: 2017

The differentiation of clenbuterol abuse and unintentional ingestion by 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 from the administration of clenbuterol from a pharmaceutical preparation and from cattle meat and liver containing residues to humans are presented. 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. Post administration urine from a controlled experiment comparing the administration of racemic clenbuterol from a registered pharmaceutical preparation and the administration of residue containing meat and liver from treated animals (non-racemic mixture) 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.

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

Maria Kristina Parr, Alexander H Schmidt

Bioanalysis 6 (2014) 3267-3270.

“Supercritical fluid chromatography allows fast and efficient separation of enantiomers.”

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).

UHPLC Method Development and Modelling in the Framework of Quality by Design

Imre Molnár, Alexander H. Schmidt, H-J. Rieger, J. Fekete, R. Kormany

TheColumn, April 2014

The goals in ultrahigh-pressure liquid chromatography (UHPLC) method development are to first find the best separation, second find the best column, and third find the most robust method in a multifactorial Design Space. Trial and error methods are not sufficient anymore and solid science based on Quality by Design (QbD) principles is required.

Rapid UHPLC Method Development for Omeprazole Analysis in a Quality-by-Design Framework and Method Transfer to HPLC using Chromatographic Modeling

Alexander H Schmidt, Mijo Stanic

LCGC North America, 32 , No 2 (2014) 126-148

The aim of this study was to apply quality-by-design principles to build in a more scientific and risk-based multifactorial strategy in the development of an ultrahigh-pressure liquid chromatography (UHPLC) method for omeprazole and its related impurities

In silico robustness testing of a compendial HPLC purity method by using of a multidimensional design space build by chromatography modeling–Case study pramipexole

Alexander H. Schmidt, Mijo Stanic, Imre Molnár

Journal of Pharmaceutical and Biomedical Analysis 91 (2014) 97-107

Purity testing of pramipexole is performed using an official (compendial) and harmonized method published in the European Pharmacopoeia (E.P.) and United States Pharmacopoeia (USP). According to this monograph the successful chromatographic separation of the API from impurities is achieved on a C18 column with gradient elution of an ion pairing buffer of pH 3.0 (mobile phase A) and acetonitrile (mobile phase B). Although not recommended in general, compendial methods are often adapted for purity testing of generic formulations. In this paper a novel approach to evaluate method robustness of an adapted method – prior of full method validation – is described. Based on Quality-by-Design (QbD) principles, a small number of experiments are performed, which after entering them into a chromatography modeling software allow to visualize a multidimensional “Design Space”, a region, in which changes in method parameters will not significantly affect the results as defined in the ICH guideline Q8(R2) leading to a more flexible method handling in routine analysis. For two different recommended C18 columns a multidimensional Design Space (Method Operating Design Region, MODR) was constructed to study the robustness of the adapted method with a newly developed Robustness Module. In a full factorial design the following six parameters were varied at three levels (low, nominal, high): Gradient time, temperature, pH of the aqueous eluent (A), flow rate, start- and end concentration of the organic mobile phase component (eluent B). The resulting 3^6 = 729 experiments were performed in silico from the previously constructed models for Design Space in less than 1 min and showed that the required resolution of 2.0 could not be reached in all experiments for the two columns which were recommended by the E.P. (failure rate 25% and 16%, respectively). However, by adjusting the gradient time, we were able to fulfill the requirements with a failure rate of zero…..

A QbD with Design-of-Experiments approach for development of a state-of-the-art UPLC purity method for carbamazepine

Alexander H. Schmidt, Carsten Wess

Journal of Liquid Chromatography & Related Technologies 37 (2014) 2653-2666

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 x 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 to study the relationship between chromatographic parameters (factors) and the resolution (response) between the peaks of interest. In a screening phase the factors known to have major effect in column selectivity (stationary phase, pH of the aqueous eluent, organic eluent type, gradient time and slope) are studied. In the second phase the chromatographic parameters identified to affect the resolution are 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 about the influencing factors and interactions. An operating space within the design space is established and a verification study confirms the robustness of the final method. Total analysis time is only 5 min, which is an impressive 22-fold increase in productivity in comparison to the method published in the European Pharmacopeia.

Using an innovative Quality-by-Design approach for development of a stability indicating UHPLC method for ebastine in the API and pharmaceutical formulations

Alexander H. Schmidt, Imre Molnar

Journal of Pharmaceutical and Biomedical Analysis 78-79 (2013) 65-74

A stability-indicating ultra high performance liquid chromatographic (UHPLC) method has been developed for purity testing of ebastine and its pharmaceutical formulations. Successful chromatographic separation of the API from impurities was achieved on a Waters Acquity UPLC BEH C18, 50 mm × 2.1 mm, 1.7 μm particle size column with gradient elution of 10 mM acetate buffer pH 6.2 and a mixture of acetonitrile/2-propanol (1:1) as the mobile phase. Incorporating Quality by Design (QbD) principles to the method development approach by using the chromatography modeling software DryLab®4 allows the visualization of a “Design Space”, a region in which changes to method parameters will not significantly affect the results as defined in the ICH guideline Q8 (R2). A verification study demonstrated that the established model for Design Space is accurate with a relative error of prediction of only 0.6%. The method was fully validated … The robustness of the developed method was studied by varying the six parameters: gradient time, temperature, ternary composition of the eluent, flow rate and start and end concentration of the gradient at 3 levels (+1, 0, −1). The resulting 729 experiments were performed in silico from the previously constructed model for Design Space and showed that the required resolution of 2.0 can be reached in all experiments. To prove the stability-indicating performance of the method, forced degradation (acid and base hydrolysis, oxidation, photolytic and thermal stress conditions) of ebastine was carried out. Baseline separation could be achieved for all peaks of the impurities, the degradation products and the API. Total run time was only 4 min, which is an impressive 40-fold increase in productivity in comparison to the method published in the Ph. Eur. monograph and allowed purity testing of more than 360 samples per day.

UPLC-MS/MS in support of cleaning validation studies in an cephalosporin antibiotics production facility

Alexander H. Schmidt, Mijo Stanic

G.I.T. Laboratory Journal Europe 5-6/2012

Pharmaceutical manufacturing equipment has to be cleaned after production in order to avoid cross contamination in the next batch of a different product. The effectiveness of the cleaning process should be confirmed by cleaning validation studies. An ultra-performance liquid chromatography method with tandem-MS detection (UPLC-MS/MS) was developed for the simultaneous determination of residues of the following beta-lactam ring containing cephalosporin antibiotics on swabs collected from pharmaceutical manufacturing equipment surfaces: cefuroxime axetil (R) and (S) isomers; cefuroxime, cefixime; cefaclor; cefpodoxime proxetil (R) and (S) isomers; cefalexin, cefadroxil

Transfer of USP-based HPLC Methods for Pantoprazole Sodium to UPLC – 20-fold increase in productivity

Alexander H. Schmidt

G.I.T. Laboratory Journal Europe 9-10/2010

HPLC is a commonly used analytical method for assaying and purity controlling of active pharmaceutical ingredients („API’s”) in the pharmaceutical industry. Method transfer to the latest technologies can be time-consuming and are therefore rarely performed for the improvement of validated methods. However, the transfer of established methods to a UPLC (ultra performance liquid chromatography) system can be worth the investment. In the reported case such an investment was rewarded with surprising savings in analysis time, operational costs and improved resolution. We demonstrate the successful method transfer for the analysis of pantoprazole sodium from the USP-recommended L1 column, run on a conventional HPLC system, to a sub 2 µm particle column on a UPLC system. With some small optimization changes, the final methodology reduced the analysis run time from 55 min with HPLC to just 3 min with UPLC, resulting in a 20-fold increase in throughput and a remarkable reduction in solvent consumption and waste disposal costs

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

Alexander H. Schmidt

Journal of Liquid Chromatography & Related Technologies Volume 29, Issue 11, 2006, pages 1663-1673

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 method’s 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.

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

Alexander H Schmidt

Journal of Liquid Chromatography & Related Technologies Volume 28, Issue 6, 2005, pages 871-881

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.

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

Alexander H. Schmidt, Imre Molnar

Journal of Chromatography A, 948 (2002) 51-63

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.

Use of an on-line, precolumn photochemical reactor in high-performance liquid chromatography of naphthodianthrones in Hypericum perforatum preparations

Alexander H. Schmidt

Journal of Chromatography A, Volume 987, Issues 1–2, 14 February 2003, Pages 181-187

A method has been developed for the determination of naphthodianthrones in Hypericum perforatum L. extracts and phytopharmaceutical preparations by high-performance liquid chromatography combined with on-line, precolumn photochemical conversion followed by photodiode-array detection. The chromatographic separation was performed on a C18 column under isocratic reversed-phase conditions. An on-line, precolumn photochemical reactor equipped with a knitted PTFE reaction coil around a visible light source was used in order to transform the light sensitive naphthodianthrones, protohypericin and protopseudohypericin, very easily into the non-protoforms, hypericin and pseudohypericin, respectively. Two UV chromatograms (photochemical reactor “on” and “off”) were compared and were quite useful in characterizing the sample. Validation studies demonstrated that this HPLC method is simple, rapid, reliable and reproducible. The time-consumptive manual irradiation of the samples is omitted by this automated on-line irradiation step. The developed method was successfully applied to the quality control of Hypericum perforatum L. extracts and its phytopharmaceutical preparations.

Fast HPLC for quality control of Harpagophytum procumbens by using a monolithic silica column: method transfer from conventional particle-based silica column

Alexander H. Schmidt

Journal of Chromatography A, Volume 1073, Issues 1–2, 6 May 2005, Pages 377-381

The applicability of a monolithic C18-bonded silica column for the rapid HPLC separation of ingredients in medicinal plants and their phytopharmaceutical preparations has been evaluated in the author’s laboratory. In this presentation, an existing method for the determination of the iridoid glycoside harpagoside in Harpagophytum procumbens (Devil’s Claw) was successfully transferred from a conventional particle-based C18 silica column to a monolithic silica column. The very high porosity of the stationary phase allows chromatography with a much lower backpressure than on conventional columns. Therefore, the flow rate could be easily increased from 0.8 mL/min (particle-based column) to 5 mL/min (monolithic column) and the run-time reduced from 30 to 5 min (that is a reduction about 85%!), without losing any chromatographic resolution of the compound of interest. The amount of harpagoside was measured with the original method on a conventional particle-based silica column and on the adapted method on a monolithic silica column. The statistical mean t-test showed no significant differences of the variances and the means indicating that the fast HPLC method is an acceptable alternative. The shorter analysis time makes the method very valuable for commercial quality control of Harpagophytum extracts and its pharmaceutical preparations.

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

Alexander H. Schmidt

Journal of Liquid Chromatography & Related Technologies Volume 28, Issue 15, 2005, pages 2339-2347

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.