High throughput multidimensional liquid chromatography approach for online protein removal and characterization of polysorbates and poloxamer in monoclonal antibody formulations.

The majority of commercially available monoclonal antibody (mAb) formulations are stabilized with one of three non-ionic surfactants: polysorbate 20 (PS20), polysorbate 80 (PS80), or poloxamer 188 (P188). All three surfactants are susceptible to degradation, which can result in functionality loss and subsequent protein aggregation or free fatty acid particle formation. Consequently, quantitative, and qualitative analysis of surfactants is an integral part of formulation development, stability, and batch release testing. Due to the heterogeneous nature of both polysorbates and poloxamer, online isolation of all the compounds from the protein and other excipients that may disturb the subsequent liquid chromatography with charged aerosol detection (LC-CAD) analysis poses a challenge. Herein, we present an analytical method employing LC-CAD, utilizing a combination of anion and cation exchange columns to completely remove proteins online before infusing the isolated surfactant onto a reversed-phase column. The method allows high throughput analysis of polysorbates within 8 minutes and poloxamer 188 within 12 minutes, providing a separation of the surfactant species of polysorbates (unesterified species, lower esters, and higher esters) and poloxamer 188 (early eluters and main species). Accuracy and precision assessed according to the International Council for harmonisation (ICH) guideline were 96 - 109 % and ≤1 % relative standard deviation respectively for all three surfactants in samples containing up to 110 mg/mL mAb. Subsequently, the method was effectively applied to quantify polysorbate 20 and polysorbate 80 in nine commercial drug products with mAb concentration of up to 180 mg/mL.

Fast and simplified quantitative multiresidue analytical method for pesticides in surface waters by UHPLC-MS/MS with online sample preparation.

A quantitative multiresidue analytical method for the simultaneous analysis of current-use agricultural pesticides in surface waters is reported. The method involves minimal sample manipulation and small sample collection volumes (for 1 mL and 5 mL injections) with online sample clean-up and analyte preconcentration on a hydrophilic-lipophilic balance (HLB) column. To our knowledge, this online approach with the use of an HLB column has not yet been reported for multiresidue pesticide analysis in surface waters. Chromatographic separations of isomeric pesticides were achieved through the sequential coupling of C8 and polar endcapped C18 analytical columns. High resolution accurate mass (HRAM) quadrupole Orbitrap spectrometry was performed in full scan mode followed by data-dependent MS/MS fragmentation (FS-ddMS2) with concurrent electrospray ionization in both positive and negative modes. The method was validated for thirty-one (31) diverse current-use pesticides and demonstrated strong linearity (R2 > 0.9912) and precision (% RSD <8.4%) with low quantitation limits (average LOQ of 41 ng L-1). The majority of target analytes experienced minimal matrix effects (<±20%) in fortified environmental water samples. When applied to surface water samples, the method detected fourteen of the target analytes, including twelve herbicides, one insecticide, and one fungicide. This method offers a fast, simple, and reliable approach for the quantitative analysis of diverse current-use pesticides in surface water samples within hours of sample collection in the field. The robust nature of the method may allow for potential application to other types of water and the targeted or untargeted screening of other emerging contaminants.

Online Extraction Followed by LC-MS/MS Analysis of Lipids in Natural Samples: A Proof-of-Concept Profiling Lecithin in Seeds.

Sample preparation is usually a complex and time-consuming procedure, which can directly affect the quality of the analysis. Recent efforts have been made to establish analytical methods involving minimal sample preparation, automatized and performed online with the analytical techniques. Online Extraction coupled with Liquid Chromatography-Mass Spectrometry (OLE-LC-MS) allows a fully connected extraction, separation, and analysis system. In this work, the lecithin profile was investigated in commercial sunflower, almonds, peanuts, and pistachio seeds to demonstrate that the concept of extraction, followed by the online analysis of the extract, could be applied to analyze this class of analytes in such complex solid matrices without a prior off-line solvent extraction step. The extraction phase gradient method was optimized. Two different analytical columns were explored, one being a conventional C18 (50 × 2.1 mm, 1.7 µm SPP) and the other a novel self-packed SIGO-C18ec (100 × 0.5, 5 µm FPP), which resulted in better separation. The analysis repeatability was investigated, and suggestions to improve it were pointed out. A characteristic ion with a m/z of 184, related to lysophosphatidylcholine structure, was used to identify the lecithin compounds. The temperature effect on the chromatograms was also explored. In short, it was found that the OLE-LC-MS approach is suitable for the analysis of lecithin compounds in seeds, being a promising alternative for lipidomics approaches in the near future.

Online biological sample preparation with restricted access hybrid carbon nanotubes for determination of anti-smoking drugs.

Untreated samples were injected directly into a column switching system, an online SPE technique, using an extraction column packed with restricted access hybrid carbon nanotubes (RAHCNTs), a novel type of restricted access material, in an ultra-high performance liquid chromatography, coupled to a mass spectrometer (UHPLC-MS/MS). The synthesis of used restricted access material was relatively simple, quick, and reproducible, and had a high material yield. Compared to its predecessor, which is covered with bovine serum albumin (Restricted Access Carbon Nanotubes-RACNTs), RAHCNTs have improved performance when used for the analysis of organic compounds. These molecules have a greater adsorption capacity due to the insertion of hydrophilic monomers (tetraethyl orthosilicate (TEOS), 3-(trimethoxysilyl)propyl methacrylate (MPS), glycerol dimethacrylate (GDMA), and hydroxyethyl methacrylate (HEMA)) in the external layer. In addition, the formation of the hybrid material provides greater chemical and thermal stability, supporting wide pH and temperature ranges, and high concentrations of acidic and basic solutions. It also supports high proportions of organic solvents in the medium. Another significant advantage of the material is its longer lifetime, as it can be reused for approximately 500 analytical cycles without any loss of efficiency, versus 300 for RACNTs. In the method developed to determine anti-smoking drugs (varenicline and bupropion) simultaneously, as well as nicotine and some of their metabolites in human blood serum, the RAHCNTs were capable of retaining the analytes efficiently, whereas the macromolecules were excluded (almost 100%). The method was linear for all the determined analytes (coefficients of determination higher than 0.99), with limits of detection and quantification ranging from 0.6 to 2.5 µg L-1 and from 1.0 to 5.0 µg L-1, respectively. High extraction recovery values were obtained (higher than 88%), as well as inter and intra-assay accuracy and precision results that are in accordance with values recommended by the FDA. The method is promising for therapeutic monitoring and new personalized strategies for patients under antismoking treatment, using a small sample volume (100 µL). In addition, RAHCNTs are capable of simultaneously extracting analytes with very different physical-chemical characteristics.

Fast automated method for the direct determination of total antimony in grape juice samples by hydride generation and atomic fluorescence spectrometric detection without external pretreatment.

Matrix complexity of fruit juices and their low antimony level requires sensitive, cost-effective instruments, time-consuming and error-prone sample pretreatment methods. Therefore, a flow-batch procedure (HG-FBA-AFS) was developed for the fast and sensitive determination of total inorganic Sb in grape juice samples by hydride generation atomic fluorescence spectrometry. The sample pretreatment, pre-reduction and stibine formation steps run through the mixing chamber. The HCl and NaBH4 concentrations, and carrier gas flowrate were optimized through a Box-Behnken design. The detection limit (LOD) was 20 ng L-1, intra and inter-day precision ranged in 3.0 - 3.5 %, and low errors within (- 2.4 to 6.6 %) for samples containing 1.23 - 4.58 µg L-1 total Sb. Both HG-FBA-AFS and reference method agreed at 95% confidence level. An 87 h-1 sample throughput, and a 1.15 mL total waste per determination attest that HG-FBA-AFS is a fast, and ecofriendly tool for determining Sb in grape juices.

Online sample pretreatment for analysis of decomposition products in lithium ion battery by liquid chromatography hyphenated with ion trap-time of flight-mass spectrometry or inductively coupled plasma-sector field-mass spectrometry.

Lithium ion batteries are essential power sources for mobile electronic devices like cell phones, tablets and increasingly used in the field of electromobility and energy transition. The commonly applied liquid electrolytes in commercial cells contain a conducting salt at relatively high concentration (LiPF6, ≥1 mol/L). For analytical battery electrolyte investigations, it is necessary to protect the column and mass spectrometer from salt precipitation and clogging. Thus, dilution of the sample is necessary which results in higher limits of detection and limits of quantification. In this study, a comprehensive online sample preparation approach for reversed phase liquid chromatography with an online-solid phase extraction was developed, which allows higher injections volumes and lower dilution factors. For the method development of the online-solid phase extraction, pristine electrolytes were used with trimethyl phosphate and triethyl phosphate as model substances for organo(fluoro)phosphates with weak and strong retention on the extraction column. Organo(fluoro)phosphates are potential hazardous decomposition products, due to their structural similarity to chemical warfare agents like sarin, and therefore their quantification is beneficial for toxicological assessment. The optimization of chromatographic parameters was performed using electrochemically aged electrolytes. For substance independent quantification with a plasma-based technique, an isocratic separation method was implemented. Using optimized conditions, LiPF6 could be removed quantitatively and the injection volume was increased up to a factor of 50, while the dilution factor could be decreased up to a factor of ten. Eleven different organo(fluoro)phosphates with an overall concentration of 133 mg/kg were found. Therefore, limit of detection and limit of quantification were improved significantly (LOQ: ≤100 µg kg-1 phosphorus content, LOD: ≤35 µg kg-1 phosphorus content). In summary, a fast online sample preparation for liquid chromatographic investigations of lithium ion battery electrolytes was implemented, validated on electrochemically aged lithium ion battery electrolyte.

Supercritical fluid extraction-supercritical fluid chromatography of saliva: Single-quadrupole mass spectrometry monitoring of caffeine for gastric emptying studies†.

Saliva is an attractive sampling matrix for measuring various endogenous and exogeneous substances but requires sample treatment prior to chromatographic analysis. Exploiting supercritical CO2 for both extraction and chromatography simplifies sample preparation, reduces organic solvent consumption, and minimizes exposure to potentially infectious samples, but has not yet been applied to oral fluid. Here, we demonstrate the feasibility and benefits of online supercritical fluid extraction coupled to supercritical fluid chromatography and single-quadrupole mass spectrometry for monitoring the model salivary tracer caffeine. A comparison of 13 C- and 32 S-labeled internal standards with external standard calibration confirmed the superiority of stable isotope-labeled caffeine over nonanalogous internal standards. As proof of concept, the validated method was applied to saliva from a magnetic resonance imaging study of gastric emptying. After administration of 35 mg caffeine via ice capsule, salivary levels correlated with magnetic resonance imaging data, corroborating caffeine's usefulness as tracer of gastric emptying (R2  = 0.945). In contrast to off-line methods, online quantification required only minute amounts of organic solvents and a single manual operation prior to online bioanalysis of saliva, thus demonstrating the usefulness of CO2 -based extraction and separation techniques for potentially infective biomatrices.

A fully validated high-throughput liquid chromatography tandem mass spectrometry method for automatic extraction and quantitative determination of endogenous nutritional biomarkers in dried blood spot samples.

Dried blood spot (DBS) sampling demonstrates multiple advantages over traditional venous blood collection in terms of quantifying biomarkers for clinical applications. The process is more convenient, less invasive and requires smaller sample size. More importantly, it lowers risk of infection and allows easier sample transportation and storage. In this study, an automated high-throughput DBS-LC-MS/MS method was developed for quantifying endogenous biomarkers in DBS (or 20 µL whole blood) and later applied in riboflavin (i.e. vitamin B2) quantification. The method consists of four steps, including internal standard spraying, high pressure sample extraction, LC-MS/MS sample analysis and automatic extraction module cleaning. The last two steps overlap, thus reducing sample preparation time and shorten the sample analysis cycle to five minutes per sample. The method was validated to be selective and sensitive (LLOQ=2 ng/mL) over a range of 2-120 ng/mL. Matrix effect was compensated by the application of internal standard, while within-run precision, between-run precision, accuracy, stability and ruggedness of the developed method were all assessed to be satisfactory. Quantitative analysis of riboflavin in 133 whole blood samples using the developed method demonstrated strong correlation compared with those quantified using traditional manual sample preparation followed by LC-MS/MS analysis (R = 0.9774). In conclusion, an automated high-throughput DBS-LC-MS/MS method was developed and validated to be sensitive, accurate and robust, suggesting great potential in the quantification of endogenous biomarkers in blood or other biofluids.

An automatic online solid-phase dehydrate extraction-ultra-high performance supercritical fluid chromatography-tandem mass spectrometry system using a dilution strategy for the screening of doping agents in human urine.

An automatic online solid-phase dehydrate extraction (SPDE)-ultra-high performance supercritical fluid chromatography (UHPSFC)-MS/MS system was developed in this study, in which the automatic SPDE procedure was coupled with UHPSFC to allow UHPSFC to analyze aqueous samples directly. Moreover, a pre-column dilution strategy was employed, which focused the analytes in strong desorption solvent on the column head and helped to obtain narrow and symmetric peaks. The online SPDE-UHPSFC-MS/MS system was firstly applied to the screening of 45 prohibited substances in human urine for doping control, during which all the mechanisms and features of the online system were fully studied. The majority (91%) of the target compounds achieved weak matrix effects (80-120%), indicating that the online method was accurate and reliable thanks to the SPDE procedure and efficient UHPSFC separation. Owing to the reduction of the matrix effects, large volume injection and the pre-column dilution, the online system could achieve high sensitivity with the LODs ranging from 0.0380 ng L-1 to 1.24 µg L-1. Under the optimized conditions, the extraction recoveries of 66% target analytes were more than 50%. All the target compounds showed good linearity with linear correlation coefficients higher than 0.9928. The accuracy values of all the spiked prohibited substances were within 80.8-119.7%, while the RSDs% for the intra-/inter-day precision were within 10.8% and 15.4%. Compared with the dilute-and-shoot-ultra-high performance liquid chromatography-MS/MS method, in which the urine samples were simply diluted before analyzing, this online method was superior in sensitivity and reducing matrix effects, which demonstrated its utility in doping control. Compared with the previously reported online SPE-SFC system, the online SPDE-UHPSFC-MS/MS system showed advantages in automation, efficiency, sensitivity and chromatographic performance. In summary, the online SPDE-UHPSFC-MS/MS system is capable of analyzing complex aqueous samples.

Recent advances in sample preparation techniques in China.

Sample preparation is the procedure before instrumental analysis and significant to its effectiveness and efficiency. However, this procedure is usually time-consuming, labor intensive, and prone to error. In the last decade, the development of sample preparation techniques has received increasing attention, especially in complex sample application. To pretreat samples faster and more effectively, advanced materials, instrumentation, and methods have been combined with typical techniques, including extraction, membrane separation, and chemical conversion techniques. Researchers in China focused on the development of simple, efficient sample preparation methods with selective enrichment and rapid separation capabilities for target analysis in complicated sample matrix and contribute almost a half of the publications in this specific field. In this review, a panorama of sample preparation techniques in China has been composed from more than 140 references, and we highlight some promising methods developed during recent years and introduce different separation materials with respect to these methods.

Online extraction of antihypertensive drugs and their metabolites from untreated human serum samples using restricted access carbon nanotubes in a column switching liquid chromatography system.

A novel analytical method was developed to determine 5 antihypertensive drugs of different pharmacological classes (angiotensin-converting enzyme inhibitors, calcium channel blockers, α-2 adrenergic receptor agonists, angiotensin II receptor blockers, and aldosterone receptor antagonists) and some of their metabolites in human serum. The untreated samples were directly analyzed in a column switching system using an extraction column packed with restricted access carbon nanotubes (RACNTs) in an ultra-high performance liquid chromatography coupled to a mass spectrometer (UHPLC-MS/MS). The RACNTs column was able to exclude approximately 100% of proteins from the samples in 2.0min, maintaining the same performance for about 300 analytical cycles. The method was validated in accordance with Food and Drug Administration (FDA) guidelines, being linear for all the determined analytes in their respective analytical ranges (coefficients of determination higher than 0.99) with limits of detection (LODs) and quantification (LOQs) ranging from 0.09 to 10.85µgL-1 and from 0.30 to 36.17µgL-1, respectively. High recovery values (88-112%) were obtained as well as suitable results for inter and intra-assay accuracy and precision. The method provided an analytical frequency of 5 samples per hour, including the sample preparation and separation/detection steps. The validated method was successfully used to analyze human serum samples of patients undergoing treatment with antihypertensive drugs, being useful for pharmacometabolomic, pharmacogenomic, and pharmacokinetic studies.

Hydrophilic interaction liquid chromatography tandem mass spectrometry analysis of malonyl-coenzyme A in breast cancer cell cultures applying online solid-phase extraction.

Cofactors such as coenzyme A and its derivatives acetyl-coenzyme A and malonyl-coenzyme A are involved in many metabolic pathways. Due to trace level concentrations in biological samples and the high reactivity of cofactors, a fast, sensitive, and selective method for quantification is mandatory. In this study, online solid-phase extraction was coupled successfully to hydrophilic interaction liquid chromatography with tandem mass spectrometry for isolation of analytes in complex matrix and quantification by external calibration. Online solid-phase extraction was carried out by application of a weak anion-exchange column, whereas hydrophilic interaction liquid chromatography separation was performed on an amide modified stationary phase. Sample preparation of the extracts before the analysis was reduced to a centrifugation and dilution step. Moreover, the applied online solid-phase extraction significantly reduced matrix effects and increased the signal-to-noise ratio. The limit of detection and the limit of quantification were in the lower nanomolar range. Finally, the applicability of this method was demonstrated on MCF-7 breast cancer cell cultures, a commonly used model system, where acetyl-coenzyme A and malonyl-coenzyme A were determined using standard addition procedure in concentrations of 1.98 µM and 41 nM, respectively.

Prototype of an Interface for Hyphenating Distillation with Gas Chromatography and Mass Spectrometry.

Chemical analysis of complex matrices-containing hundreds of compounds-is challenging. Two-dimensional separation techniques provide an efficient way to reduce complexity of mixtures analyzed by mass spectrometry (MS). For example, gasoline is a mixture of numerous compounds, which can be fractionated by distillation techniques. However, coupling conventional distillation with other separations as well as MS is not straightforward. We have established an automatic system for online coupling of simple microscale distillation with gas chromatography (GC) and electron ionization MS. The developed system incorporates an interface between the distillation condenser and the injector of a fused silica capillary GC column. Development of this multidimensional separation (distillation-GC-MS) was preceded by a series of preliminary off-line experiments. In the developed technique, the components with different boiling points are fractionated and instantly analyzed by GC-MS. The obtained data sets illustrate dynamics of the distillation process. An important advantage of the distillation-GC-MS technique is that raw samples can directly be analyzed without removal of the non-volatile matrix residues that could contaminate the GC injection port and the column. Distilling the samples immediately before the injection to the GC column may reduce possible matrix effects-especially in the early phase of separation, when molecules with different volatilities co-migrate. It can also reduce losses of highly volatile components (during fraction collection and transfer). The two separation steps are partly orthogonal, what can slightly increase selectivity of the entire analysis.

Rhamnolipid biosurfactant analysis using online turbulent flow chromatography-liquid chromatography-tandem mass spectrometry.

Rhamnolipids are biosurfactants produced by a variety of bacterial species that present a promising alternative to surfactants from petrochemical or oleochemical origin. The success of the fermentation is evaluated by subsequent qualitative and quantitative analysis. However, the sample preparation for high numbers of samples is often laborious and inefficient. In this study an online sample preparation is developed for the qualitative and quantitative analysis of rhamnolipids by LC-MS/MS. Online sample preparation is carried out on a TurboFlow Cyclone MAX column using turbulent flow chromatography. Sample preparation prior the analysis is minimized to a dilution and syringe filtration step leading to an instrumental analysis time of 33min. The limit of detection and the limit of quantification were 0.4ng and 0.6ng on column, respectively. Recovery of the main mono- and di-rhamnolipids from a fermentation sample was 102-104%. Additionally, the rhamnolipid biosynthetic precursors 3-hydroxy(alkanoyloxy)alkanoic acids (HAAs) are covered, albeit extraction is not quantitative (85-90%). The analysis of rhamnolipids from four different microbial species was in good agreement with previous reports. The presented method allows rapid and comprehensive analysis of rhamnolipids with minimal sample preparation directly from the fermentation broth. The application of complementary data-dependent MS/MS acquisition enables non-target screening of rhamnolipids.

Rapid and direct analysis of statins in human plasma by column-switching liquid chromatography with restricted-access material.

This study presents the development of a column-switching liquid chromatographic method with direct injection of human plasma for simultaneous determination of four statins (lovastatin, pravastatin, rosuvastatin and simvastatin), the main class of drugs used in the treatment of hyperlipidemia. By using a C18 (30 mm × 4.6 mm, 15 µm) a lab made bovine serum albumin restricted access material (RAM) column was prepared and compared with a commercial alquil-diol silica RAM column (C18, 25 mm × 4.0 mm, 25 µm) in terms of their protein exclusion capacity and micromolecules retention. Foreflush and backflush modes were compared for both RAM columns to the number of theoretical plates, asymmetry, resolution and chromatographic run time. The developed method was validated in the range from 125 to 876 ng mL(-1) for lovastatin, rosuvastatin and simvastatin, and from 500 to 2000 ng mL(-1) for pravastatin, presenting selectivity, precision and accuracy intra and inter-run. Total analysis time (sample preparation and chromatographic separation) was only 16 min when the backflush mode was employed in the column-switching system.