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Advancements in column technology resulted in smaller particles and more efficient phases. In parallel, the use of columns with reduced dimensions is becoming more common. This means the effective column volume is also decreased, thereby making the systems more susceptible to effects of band broadening due to extra-column volume. Despite these trends and the fact that a growing number of miniaturized liquid chromatography systems are being offered commercially, manufacturers often stick to the modular concept with dedicated units for pumps, column oven, and detectors. This modular design results in long connection capillaries, which leads to extra-column band broadening and consequently prevents the exploitation of the intrinsic efficiency of state-of-the-art columns. In particular, band broadening post column has a considerable negative effect on efficiency. In this study, mass flow and concentration-dependent detectors were examined for their influence on band broadening using a micro-LC system. A mass spectrometric detector, an evaporative light scattering detector, two UV detectors, and a previously undescribed fluorescence detector were compared. The influence on efficiency is compared using plate height vs linear velocity data and peak variance. It is shown that an increase in the inner diameter after the post-column transfer capillary leads to significant loss in plate height. Comparing the UV detectors, it could be shown that the dispersion was reduced by 38% by the reduction of the post-column volume. The largest variance was found for the evaporative light scattering detector, which was 368% higher compared to the variance of the detector with the least effect on band broadening.

In order to ensure a high level of product quality and safety, regular quality controls are mandatory, especially in the pharmaceutical industry. These quality controls are strictly regulated and require a high level of documentation. With the goal of complete traceability, these regulations are constantly being tightened, while a majority of laboratories are working still completely paper-based. This leads to an ever-increasing workload that keeps laboratory staff away from value-adding analytical work. In order to realize complete traceability, a reduction in documentation errors and at the same time a reduction of the individual workload, the digitization of complete workflows seems to be a promising solution.Due to the ongoing shortage of IT specialists and the resulting high implementation costs, many laboratories are understandably hesitant. In this chapter an alternative is presented on how to approach the digitization of complete workflows without the need for IT specialists. The example of quality control analysis of cytotoxic drug solutions was chosen to demonstrate the challenges of such a digitization project. In this way, we contribute to a comprehensive understanding of the tools already available, which can also help other laboratories in their digitization efforts. At the end compliance with GMP and EN ISO/IEC 17025 (2017) regulations was reached.

Due to the complex manufacturing process of therapeutic monoclonal antibodies, it is hardly possible to produce an identical copy of the original product (originator). Consequently, follow-on products (biosimilars) must demonstrate their efficacy being similar to the originator in terms of structure and function. During this process, a variety of analytical methods are required for this purpose. This study focuses on three particularly relevant analytical techniques: high-resolution mass spectrometry, fragment crystallisable (Fc) affinity chromatography, and two-dimensional peptide mapping. Each analytical method proved able to identify specific differences between originator and biosimilar. High-resolution mass spectrometry was used to characterize the glycan pattern. It was shown that a trastuzumab biosimilar did not have the G0:G0F sugar modification identified in the originator. The application of affinity chromatography to rituximab showed that originator and biosimilar interacted differently with the immobilized Fc receptor. Furthermore, 2D-HPLC peptide mapping demonstrated the influence of orthogonality of separation dimensions, leading to differentiation of a rituximab originator and biosimilar.

Regarding endocrine disrupting chemicals, an impact on aquatic organisms at already very low concentrations is observed. Consequently, there is a need for sensitive analytical methods. Currently, no instrumental analysis methods exist that fulfil this requirement. To overcome these limitations, effect-based methods (EBMs) are one option for screening and quantification. Determination of agonistic effects caused by estrogens are the focus of current research activities due to environmental impacts and regulatory measures. However, antagonistic effects have to be considered to be able to correlate effects to single compounds, which is still necessary due to regulatory requirements. Since not much is known about relevant antagonists, this review highlights the perspectives and limitations of EBMs. The relevance of antagonistic acting compounds shows the potential of a systematic identification by effect-directed analysis. Collected data of already known antagonists are presented and discussed in a harmonized fashion as basis for future studies.

The drugs used for treatment during chemotherapy are manufactured individually for each patient in specialised pharmacies. Thorough quality control to confirm the identity of the delivered active pharmaceutical ingredient and the final concentration of the prepared application solution is not standardized yet except for optical or gravimetric testing. However, solution stability problems, counterfeit drugs, and erroneous or deliberate underdosage may occur and negatively influence the quality of the product and could cause severe health risks for the patient. To take a step towards analytical quality control, an on-site analytical instrument using Raman and UV absorption spectroscopy was employed and the results were compared to high-performance liquid chromatography coupled to diode array detection. Within the scope of the technology evaluation, the uncertainty of measurement was determined for the analysis of the five frequently used cytostatic drugs 5-fluorouracil, cyclophosphamide, gemcitabine, irinotecan and paclitaxel. The Raman/UV technique (2.0–3.2% uncertainty of measurement; level of confidence: 95%) achieves a combined uncertainty of measurement comparable to HPLC-DAD (1.7–3.2% uncertainty of measurement; level of confidence: 95%) for the substances 5-fluorouracil, cyclophosphamide and gemcitabine. However, the uncertainty of measurement for the substances irinotecan and paclitaxel is three times higher when the Raman/UV technique is used. This is due to the fact that the Raman/UV technique analyses the undiluted sample; therefore, the sample has a higher viscosity and tendency to foam. Out of 136 patient-specific preparations analysed within this study, 96% had a deviation of less than 10% from the target content.

Complete characterization and quantification of monoclonal antibodies often rely on enzymatic digestion with trypsin. In order to accelerate and automate this frequently performed sample preparation step, immobilized enzyme reactors (IMER) compatible with standard HPLC systems were used. This allows an automated online approach in all analytical laboratories. We were able to demonstrate that the required digestion time for the model monoclonal antibody rituximab could be reduced to 20 min. Nevertheless, a previous denaturation of the protein is required, which also needs 20 min. Recoveries were determined at various concentrations and were 100% ± 1% at 100 ng on column, 96% ± 7% at 250 ng on column and 98% ± 2% at 450 ng on column. Despite these good recoveries, complete digestion was not achieved, resulting in a poorer limit of quantification. This is 50 ng on column under optimized IMER conditions, whereas an offline digest on the same system achieved 0.3 ng on column. Furthermore, our work revealed that TRIS buffers, when used with an IMER system, led to alteration of the peptides and induced modifications in the peptides. Therefore, the addition of TRIS should be avoided when working at elevated temperatures of about 60 °C. Nevertheless, our results have shown that the recovery is not significantly influenced whether TRIS is used or not (recovery: 96 ± 7% with TRIS vs. 100 ± 9% without TRIS).

The very fast separation and identification of isomeric small substances with a molecular weight under 800 Da is still a challenge for high-throughput analysis. Inadequate chromatographic or spectrometric separation hampers an unequivocal identification of isomers, which may be recorded as a sum parameter. Reversed-phase liquid chromatography can be considered a generic method for the separation of isomers. However, a separation is usually achieved within minutes and not milliseconds as is typical for ion mobility spectrometry. The aim of this study, therefore, was to investigate the potential of IMS to separate small isomeric compounds. Hence, 23 substances divided into 11 isomeric groups have been selected. Among them, cancer drugs, hormones, pain relievers and others are contained. Three ion mobility spectrometers with different separation principles were compared with respect to their resolving performance. These systems comprised a traveling wave ion mobility spectrometer, a differential ion mobility spectrometer and a differential mobility analyzer. For reference, the chromatographic resolution and peak capacity was determined by high-performance liquid chromatography using a reversed phase.

Many gas chromatography-flame ionization detection (GC/FID) studies are dealing with response behavior of analytes such as alcohols and alkanes. Studies in the field of liquid chromatography (LC)/FID mainly focused on volatile analytes. In contrast, studies on LC/FID by conveyor type interface covered high molecular weight non-volatile biopolymers, whereby no response factors were calculated. With this study, we fill the gap and present response factors of volatile and non-volatile analytes by LC/FID in terms of flow injection (FIA) measurements of the single compounds without an analytical separation by an LC column. In the present study, 56 different compounds such as carboxylic acids, N-heterocycles, halogenated acids, pharmaceuticals, and other compounds were investigated. In some cases, the obtained response factor data confirmed aspects known from GC/FID studies. But this study also disproves several assumptions done in previous response studies as well as the prediction models based upon the experimental data and literature. Especially the response factors and effective carbon number (ECN) values of structural isomers such as pyrazine, pyridazine, and pyrimidine are assumed to be equal in current response prediction models. Contradictory to these assumptions, the experimental response factors and ECN values of, e.g., the structural isomers pyrazine (RFExp = 0.59; ECNExp = 3.66), pyridazine (RFExp = 0.66; ECNExp = 4.1), and pyrimidine (RFExp = 0.63; ECNExp = 3.93) reveal different experimental response factors and ECN than proposed by response factor prediction models (RFExp = 0.64; ECNExp = 4). Graphical abstract Graphical abstract.

Within this study, a novel liquid chromatography (LC)/flame ionization detector (FID) interface was improved. In contrast to previously published interface concepts, the main nebulizer body and the transfer capillary was made of stainless steel. Previously reported problems such as blocking of the transfer capillary were investigated. The simple design of the here presented nebulizer interface allows a convenient handling and the exchangeability of all nebulizer parts targets fast maintenance during routine analysis. A significant advantage is the capability to implement the novel interface into most common gas chromatography (GC)/FID systems. The effects of the instrumental parameters such as backpressure, gas flow, distance between nebulizer nozzle and FID collector or FID temperature on the signal were analyzed and optimized. The influence of the nebulizer material on flame stability and capillary blockage, a well-known problem of former coupling systems, was investigated, too. Finally, the novel interface was validated for the analysis of selected compounds known from literature. Obtained results for chromatographic separation of the alcohols propanol, butanol, pentanol and hexanol, used within previous studies in the field of LC/FID coupling are shown. Limits of detection (LODs), sensitivity and linearity found within this work are compared with LC/FID interfaces developed in the past.

Background Patient-specific preparations have become a central therapy concept in oncological treatment. The highly potent cytostatic agents are characterised by a narrow therapeutic range. Therefore, exact dosage is important, as lower amounts reduce the effectiveness and higher doses increase the risk of severe side effects. Compound confusion can even result in fatal consequences. Missing dependent and independent controls regarding concentration and identity pose a risk for patient safety. Purpose We developed a concept for a two-stage quality control of the infusion solutions. Drug identity and concentration can be checked onsite after preparation using a combined UV- and Raman spectrometer (UV-Raman). This is complemented by an independent method using liquid chromatography coupled to UV detection (HPLC-UV). Material and methods Methods for the analysis of seven cytostatic drugs and two monoclonal antibodies were developed and validated on an i-QCRx UV-Raman system (B and W Tek Europe GmbH, Lubeck, Germany) and on an Agilent 1200 series HPLC-UV system (Agilent Technologies, Waldbronn, Germany). Sample transport and preparation were evaluated to ensure valid results. In a pilot study we analysed samples from different pharmacies in both systems. Results Method development and validation were successful for the investigated compounds in both systems. HPLC-UV is more sensitive than UV-Raman. However, due to the content of the preparations, real samples had to be diluted before applying HPLC-UV analysis. Sensitivity of the UV-Raman spectrometer fits to the required concentration range without further dilution. All methods showed reproducible results, UV-Raman varied by 0.44% in a repeated analysis (n=3) of 5-fluorouracil, while HPLC-UV varied by 0.14%. Results of the investigated samples were also equivalent. In a sample containing paclitaxel with a target concentration of 0.72 mg/mL we determined 0.73 mg/mL (101%) using UV-Raman and 0.69 mg/mL (96%) using HPLC-UV, for example. Conclusion UV-Raman and HPLC-UV are suitable for determining the content of patient-individual preparations, both with individual assets and drawbacks. The study showed that the two-stage control concept is appropriate to ensure a high-quality level for patient-individual preparations. References and/or acknowledgements No conflict of interest.

The injection volume and the associated column volume overload is one of the most common issues in miniaturized chromatography. The injection volume should not exceed 10% of the effective column volume. A further reduction of the injection volume leads to an increase in chromatographic efficiency. However, the signal intensity must be above a certain threshold to generate a chromatographic peak that can be detected. Therefore, the injection volume has to be optimized to reach the ideal balance between chromatographic efficiency and sensitivity. This study examined the general influence of the injection volume for both isocratic and gradient elution, depending on the retention factor and peak standard deviation. For this purpose, substances of different polarity were selected to represent a broad elution spectrum. Besides the model analyte naphthalene, these were mainly pharmaceuticals. For all measurements a microbore column with an ID of 300 µm and packed with 1.9 μm fully porous particles was used. For isocratic elution, the injection volume was varied between 4 and 16% of the effective column volume. The retention factors were adjusted between 2 and 10. For gradient elution, the injection volume was varied between 4 and 160% of the effective column volume. The observed effects were further investigated using the gradient kinetic plot theory. In isocratic elution, a loss in plate height up to 50% was observed for components that elute near the void time. A significant reduction of the chromatographic efficiency was noticed up to a retention factor of 4. In gradient elution, a reduction in peak capacity could only be observed if the injection volume exceeded 40% of the effective column volume. For some substances, only a slight loss in peak capacity was noticed even with a volume overload of 160%.

The efficiency of miniaturized liquid chromatography columns with inner diameters between 200 and 300 μm has been investigated using a dedicated micro-liquid chromatography system. Fully porous, core-shell and monolithic commercially available stationary phases were compared applying van Deemter and kinetic plot analysis. The sub-2 μm fully porous as well as the 2.7 μm core-shell particle packed columns showed superior efficiency and similar values for the minimum reduced plate heights (2.56-2.69) before correction for extra-column contribution compared to normal-bore columns. Moreover, the influence of extra-column contribution was investigated to demonstrate the difference between apparent and intrinsic efficiency by replacing the column by a zero dead volume union to determine the band spreading caused by the system. It was demonstrated that 72% of the intrinsic efficiency could be reached. The results of the kinetic plot analysis indicate the superior performance of the sub-2 μm fully porous particle packed column for ultra-fast liquid chromatography.

Forderungen nach hoherer Geschwindigkeit und die Komplexitat der Fragen geben der analytischen Chemie neue Impulse. So nutzt die Prozessanalytik gepulste Ramanspektrometer, die Elemente Arsen und Quecksilber dominieren die Forschung in der Speziesanalytik, und die Omics-Techniken entwickeln sich zu Multi-Omics-Ansatzen. Ambiente MS-Techniken benotigen keine aufwendige Probenvorbereitung, multidimensionale Trenntechniken werden verstarkt in der Routine eingesetzt, und Chip-basierte Trennungen fallen durch Schnelligkeit auf. Molekulspektroskopie und Massenspektrometrie dominieren die bildgebenden Verfahren, und die Lateralauflosung der ToF-Sekundarionenmassenspektrometrie hat sich bei Oberflachenuntersuchungen verbessert.

Monoclonal antibodies are a group of commonly used therapeutics, whose occupational health risk is still discussed controversially. The long-term low-dose exposure side effects are insufficiently evaluated; hence, discussions are often based on a theoretical level or extrapolating side effects from therapeutic dosages. While some research groups recommend applying the precautionary principle for monoclonal antibodies, others consider the exposure risk too low for measures taken towards occupational health and safety. However, both groups agree that airborne monoclonal antibodies have the biggest risk potential. Therefore, we developed a peptide-based analytical method for occupational exposure monitoring of airborne monoclonal antibodies. The method will allow collecting data about the occupational exposure to monoclonal antibodies. Thus, the mean daily intake for personnel in pharmacies and the pharmaceutical industry can be determined for the first time and will help to substantiate the risk assessment by relevant data. The introduced monitoring method includes air sampling, sample preparation and detection by liquid chromatography coupled with high-resolution mass spectrometry of individual monoclonal antibodies as well as sum parameter. For method development and validation, a chimeric (rituximab), humanised (trastuzumab) and a fully humanised (daratumumab) monoclonal antibody are used. A limit of detection between 1 μg per sample for daratumumab and 25 μg per sample for the collective peptide is achieved. Graphical abstract Demonstration of the analytical workflow, from the release of monoclonal antibodies to the detection as single substances as well as sum parameter.

The interest in two-dimensional liquid chromatography separations is growing every year together with the number of open questions on the benefits of multidimensional systems in comparison to one-dimensional liquid chromatography. In order to solve some of these open questions this work presents a comparison of one-dimensional and microscale two-dimensional liquid chromatography coupled to high resolution mass spectrometry for targeted analysis in wastewater. The comparison is based on the evaluation of a reference standard mixture containing 99 compounds and a real wastewater sample. For the evaluation and compound identification three different criteria were chosen. At first, a deviation of ±5 ppm from the exact mass was defined as acceptable to include the compound for further evaluation. To eliminate false positive results, a maximum retention time deviation of less than 2.5% for each compound of the reference standard and the compounds detected in the wastewater sample was defined for a positive identification as a second criterion for 1D-LC and the second dimension of 2D-LC. In the third step, fragment information from MS/MS experiments was used for further identification of compounds in wastewater. Additionally, the influence of a higher mass accuracy of 1 ppm on the number of identified compounds in comparison to a mass accuracy of 5 ppm was investigated. The results showed that the number of identified compounds was higher by a factor of three in the wastewater sample when using the microscale 2D-LC approach. Moreover, a higher reliability for compound identification is obtained when using retention time and MS/MS information as identification criteria instead of only applying high mass accuracy of 1 or 5 ppm.

The performance of micro-liquid chromatography columns with an inner diameter of 0.3mm was investigated on a dedicated micro-LC system for gradient elution. Core-shell as well as fully porous particle packed columns were compared on the basis of peak capacity and gradient kinetic plot limits. The results for peak capacity showed the superior performance of columns packed with sub-2μm fully porous particles compared to 3.0μm fully porous and 2.7μm core-shell particles within a range of different gradient time to column void time ratios. For ultra-fast chromatography a maximum peak capacity of 16 can be obtained using a 30s gradient for the sub-2μm fully porous particle packed column. A maximum peak capacity of 121 can be achieved using a 5min gradient. In addition, the influence of an alternative detector cell on the basis of optical waveguide technology and contributing less to system variance was investigated showing an increased peak capacity for all applied gradient time/column void time ratios. Finally, the influence of pressure was evaluated indicating increased peak capacity for maximum performance whereas a limited benefit for ultra-fast chromatography with gradient times below 30s was observed.

The efficiency of miniaturized liquid chromatography columns with inner diameters between 200 and 300 μm has been investigated using a dedicated micro-liquid chromatography system. Fully porous, core-shell and monolithic commercially available stationary phases were compared applying van Deemter and kinetic plot analysis. The sub-2 μm fully porous as well as the 2.7 μm core-shell particle packed columns showed superior efficiency and similar values for the minimum reduced plate heights (2.56-2.69) before correction for extra-column contribution compared to normal-bore columns. Moreover, the influence of extra-column contribution was investigated to demonstrate the difference between apparent and intrinsic efficiency by replacing the column by a zero dead volume union to determine the band spreading caused by the system. It was demonstrated that 72% of the intrinsic efficiency could be reached. The results of the kinetic plot analysis indicate the superior performance of the sub-2 μm fully porous particle packed column for ultra-fast liquid chromatography.

A fast quantification method for the determination of 11 antineoplastic drugs from wipe samples was developed using micro-scale liquid chromatography in combination with tandem mass spectrometry. The extraction efficiency from the wipes has been investigated using different extraction solvents. The results indicate that a mixture of 70/30 water/isopropanol (v/v) acidified with 0.1 % formic acid is suitable to desorb the antineoplastic drugs with sufficient recovery between 80 and 120 %. Compared to conventional liquid chromatography, the total analysis time can be reduced to 2.25 min using a 50 × 0.3 mm column at a flow rate of 25 μL min−1. Ion source parameters as well as the injection volume were optimized to ensure the highest sensitivity. The results of method validation showed an instrumental limit of quantification between 0.0068 and 0.0488 ng mL−1 using an injection volume of 4.25 μL estimated by the signal to noise ratio. Moreover, the retention time repeatability was determined with a maximum relative standard deviation of 0.4 %.

For the identification of the optimal column combinations, a comparative orthogonality study of single columns and columns coupled in series for the first dimension of a microscale two-dimensional liquid chromatographic approach was performed. In total, eight columns or column combinations were chosen. For the assessment of the optimal column combination, the orthogonality value as well as the peak distributions across the first and second dimension was used. In total, three different methods of orthogonality calculation, namely the Convex Hull, Bin Counting, and Asterisk methods, were compared. Unfortunately, the first two methods do not provide any information of peak distribution. The third method provides this important information, but is not optimal when only a limited number of components are used for method development. Therefore, a new concept for peak distribution assessment across the separation space of two-dimensional chromatographic systems and clustering detection was developed. It could be shown that the Bin Counting method in combination with additionally calculated histograms for the respective dimensions is well suited for the evaluation of orthogonality and peak clustering. The newly developed method could be used generally in the assessment of 2D separations. Graphical Abstract ᅟ.

High-temperature liquid chromatography provides several advantages concerning HPLC method development and is needed to realize special hyphenation techniques. However, hardware limitations and stationary phase degradation can prevent the successful application of this technique. Therefore, column stability is of major importance. The presented study contains results of long-term high-temperature stability tests for eight modern commercially available HPLC stationary phases. Six of them were silica-based reversed-phase C18 columns tested according to an earlier reported procedure (Teutenberg, 2009 [12]). On the basis of the extended data set that covers a wide range of column technologies, a comparative approach using retention factor analysis was evaluated in order to categorize the columns according to their long-term high-temperature stability at 150 °C and changing pH value. This approach offers a more objective alternative to conventional listings in which temperature maxima are compared for columns tested under different conditions. Additionally, customized tests are presented for one promising polymer-based C18 stationary phase and an amide column for hydrophilic interaction liquid chromatography.

Solvent compatibility is a limiting factor for the success of two-dimensional liquid chromatography (2-D LC). In the second dimension, solvent effects can result in overpressures as well as in peak broadening or even distortion. A peak shape study was performed on a one-dimensional high-performance liquid chromatography (HPLC) system to simulate the impact of peak distorting solvent effects on a reversed-phase second dimension separation operated at high temperatures. This study includes changes in injection volume, solute concentration, column inner diameter, eluent composition and oven temperature. Special attention was given to the influence of high temperatures on the solvent effects. High-temperature HPLC (HT-HPLC) is known to enhance second dimension separations in terms of speed, selectivity and solvent compatibility. The ability to minimise the viscosity contrast between the mobile phases of both dimensions makes HT-HPLC a promising tool to avoid viscosity mismatch effects like (pre-)viscous fingering. In case of our study, viscosity mismatch effects could not be observed. However, our results clearly show that the enhancement in solvent compatibility provided by the application of high temperatures does not include the elimination of solvent strength effects. The additional peak broadening and distortion caused by this effect is a potential error source for data processing in 2-D LC.

The method of high-temperature liquid chromatography isotope ratio mass spectrometry (HTLC-IRMS) is used to determine the origin or authenticity of compounds. Currently, the drawback of this hyphenation is the interface which causes pronounced band broadening due to a large extra-column volume. Therefore, the aim of this study is to determine suitable column geometries and particle sizes at different temperature and to study the effect of extra-column band broadening. The tools to assess the efficiency of columns are van Deemter and kinetic plots. By comparison of different column geometries and particle sizes, it could be shown that 3.0 mm ID columns achieve a higher performance than 2.1 mm ID columns and a particle size of 1.7 μm is advantageous over 3.5 and 5.0 μm particles when the injection volume is adjusted to 2 μL and the temperature is higher than 60 °C. Because water was the mobile phase, the retention factor could not be kept constant at different column temperatures. The lower retention factor at elevated temperatures leads to a decrease of the plate number, because of the relatively larger contribution to extra-column band broadening at lower retention factors. This is the reason why 3.0 mm ID columns should be preferred for the HTLC-IRMS hyphenation when the separation is carried out under isothermal and isocratic conditions.

Within the scope of research for target and non-target LC–MS/MS analysis of membrane degradation products of polymer electrolyte membrane fuel cells, a systematic method development for the separation of structurally similar compounds was performed by phase optimized liquid chromatography. Five different stationary phases with different selectivities were used. Isocratic separation for 4-hydroxybenzoic acid, isophthalic acid, terephthalic acid, 4-hydroxybenzaldehyde and 4-formylbenzoic acid was achieved on a C18 and a Phenyl phase. Using the PRISMA model the separation efficiency was optimized. This was achieved on a serially connected mixed stationary phase composed of 30 mm C18, 150 mm Phenyl and 60 mm C30. For the LC–MS screening of unknown degradation products from polymer electrolyte membranes in the product water of a fuel cell, a solvent gradient is mandatory for less polar or later eluting compounds. By means of 4-mercaptobenzoic acid it could be shown that a solvent gradient can be applied in order to elute later eluting compounds in a short time. The adaptability of this method for the qualitative analysis by target and non-target LC–MS/MS screening has been shown by means of 4-hydroxybenzoic acid. The combination of solvent gradient and isocratic conditions makes this approach attractive for the purpose of a screening method for known and unknown analytes in a water sample.

In this study the temperature stability of several normal phase and RP columns was investigated using a water-only mobile phase. The temperature was adjusted to 120 degrees C for the bare silica stationary phases and to 185 degrees C for the metal oxide and carbon stationary phases. It could be shown that metal oxide stationary phases exhibited excellent thermal stability over the duration of the test period and are therefore suitable for high temperature LC applications.

Optimization of the chromatographic selectivity is the most important parameter if a separation is needed for the hyphenation of liquid chromatography with mass spectrometry. In mass spectrometry, this is necessary if the investigated analytes have identical mass transitions, like isomers or epimers. For the separation of the 12 most important antineoplastic drugs, a selectivity screening was performed using 20 columns and two organic modifiers and temperatures to find a suitable phase system in order to separate critical peak pairs. Therefore, an evaluation strategy was applied in form of a principal component analysis (PCA), selectivity factor, and overall selectivity comparison to find a suitable phase system. Some boundary conditions were defined to consider the specific requirements of tandem mass spectrometry. The results clearly indicated that the selectivity factor of the critical peak pairs increased using methanol at higher temperature.

Six anticancer drugs have been eluted on a polystyrene-divinylbenzene (PS-DVB) column with buffered superheated water as the mobile phase. The temperature range studied was from ambient temperature up to 160 degrees C, and the pH of the water was adjusted to 11.5 and 3.5 with phosphate buffer. It was possible to separate the substances 5-fluorouracil (5-FU), methotrexate (MTX), 7-hydroxymethotrexate (7-OH-MTX), and etoposide (VP-16) in one chromatographic run. The separation of these substances was optimized when adjusting the pH from 11.5 to 3.5, resulting in a total elution time of less than 13 min. Furthermore, retention factors of all of the investigated substances were measured at different temperatures and pH values.

Generally, compound-specific isotope analysis of steroids is carried out by gas chromatography combined with isotope ratio mass spectrometry. Thus, a derivatization of the steroids prior to the measurement is compulsory, and a correction of the isotopic data is often necessary. To overcome this limitation, we present a new approach of high-temperature liquid chromatography coupled with photodiode array detection and isotope ratio mass spectrometry (HT-LC/PDA/IRMS) for the carbon isotope ratio analysis of unconjugated steroids. A steroid mixture containing 19-norandrosterone, testosterone, epitestosterone, androsterone, and 5β-pregnane-3α,17α,20α-triol was fully separated on a C4 column under high-temperature elution with water as the sole eluent. The accuracy for isotope analysis (±0.5 ‰) was around 20 μg g(-1) for testosterone, epitestosterone (79 ng steroid absolute on column), and 30 μg g(-1) for 19-norandrosterone, androsterone, and 5β-pregnane-3α,17α,20α-triol (119 ng steroid absolute on column). The applicability of the method was tested by measuring a pharmaceutical gel containing testosterone. With this work, the scope of LC/IRMS applications has been extended to nonpolar compounds.

In the present work it is shown that the linear elution strength (LES) model which was adapted from temperature-programming gas chromatography (GC) can also be employed for systematic method development in high-temperature liquid chromatography (HT-HPLC). The ability to predict isothermal retention times based on temperature-gradient as well as isothermal input data was investigated. For a small temperature interval of Δ T = 40 ° C , both approaches result in very similar predictions. Average relative errors of predicted retention times of 2.7% and 1.9% were observed for simulations based on isothermal and temperature-gradient measurements, respectively. Concurrently, it was investigated whether the accuracy of retention time predictions of segmented temperature gradients can be further improved by temperature dependent calculation of the parameter S T of the LES relationship. It was found that the accuracy of retention time predictions of multi-step temperature gradients can be improved to around 1.5%, if S T was also calculated temperature dependent. The adjusted experimental design making use of four temperature-gradient measurements was applied for systematic method development of selected food additives by high-temperature liquid chromatography. Method development was performed within a temperature interval from 40 °C to 180 °C using water as mobile phase. Two separation methods were established where selected food additives were baseline separated. In addition, a good agreement between simulation and experiment was observed, because an average relative error of predicted retention times of complex segmented temperature gradients less than 5% was observed. Finally, a schedule of recommendations to assist the practitioner during systematic method development in high-temperature liquid chromatography was established.