Retention Time Reproducibility Research Articles

Development of LC-FAIMS-MS and its application to lipidomics study of Acinetobacter baumannii infection

The recent advances in mass spectrometry (MS) technologies have enabled comprehensive lipid profiling in biological samples. However, the robustness and efficiency of MS-based lipidomics is compromised by the complexity of biological samples. High-field asymmetric waveform ion mobility spectrometry (FAIMS) is a technology that can continuously transmit one type of ion, independent of the mass-to-charge ratio. Here we present the development and application of LC-FAIMS-MS/MS-based platform for untargeted lipidomics. We used 3 optimally balanced compensation voltages, i.e., 29 V, 34 V and 39 V, to analyze all subclasses of glycerophospholipids. The reproducibility of the method was evaluated using reference standards. The reproducibility of retention times ranged from 0.9% to 1.5% RSD; whereas RSD values of 5%–10% were observed for peak areas. More importantly, the coupling of a FAIMS device can significantly improve the robustness and efficiency. We exploited this NPLC-FAIMS-HRMS to analyze the serum lipid profiles in mice infected intranasally with Acinetobacter baumannii. The temporal profiles of serum lipids after A. baumannii inoculation were obtained for 4 h, 8 h, and 24 h. We found that nearly all ether PC and ether PE lipids were significantly decreased 8 h after inoculation. The resultant volcano plot illustrated the distribution of 28 increased and 28 decreased lipid species in mouse sera 24 h after inoculation. We also found that a single ether PE composition can comprise multiple isomeric structures, and the relative abundance of each isomer could be quantified using the newly developed NPLC-FAIMS-PRM method. We have demonstrated that the proposed LC-FAIMS-MS is a valuable platform for lipidomics.

Development and validation of stability indicating HPLC-DAD assay of adapalene, methylparaben and propylparaben in a cosmeceutical gel matrix with blueness, greenness and whiteness assessment via relative ranking: An ab initio-sub finem juxtaposed non-traditional approaches

Adapalene (ADA) is primarily used as a cosmeceutical gel in the treatment of mild-moderate acne and is also used off-label to treat keratosis pilaris as well as other skin conditions. A stability-indicating fit-for-purpose (FFP) method has been developed for co-determination of ADA, methylparaben (MPN), and propylparaben (PPN) combined in a cosmeceutical gel dosage form. This method was based on a reversed-phase RP-HPLC separation of the cited analytes. It avoided the use of tetrahydrofuran (THF), which is commonly used by both compendial and published procedures for determination of ADA, thus improving method blueness (applicability), greenness and whiteness. The method development and validation followed non-traditional eco-friendly approaches including chemical simulations and empirical modelling. The HPLC separation was performed on an RP Zorbax Eclipse XDB-C8 analytical column [150 × 4.6 mm, 5 µm (80 Å)] with gradient elution system of HPLC grade acetonitrile and 50 mM aqueous sodium dihydrogen phosphate buffer containing 5 mM ethylenediaminetetraacetic acid (EDTA) adjusted to pH 8.0 as the mobile phase at a flow rate of 2.5 mL min−1 and column oven was set at 25 °C. EDTA played an important role for reproducibility of retention time of the three analytes especially for ADA. It controlled deprotonation of the three acidic analytes under study and hence reproduce their ionization, hydrophobicity, and hence retention time. Quantitation was achieved with the photodiode array detection (DAD) at wavelengths of 256 nm for both MPN and PPN and 312 nm for ADA. Target Measurement Uncertainty (TMU) for the three analytes was determined via two approaches; misclassification rates approach employing 100,000 Monte Carlo simulations (MCS) and in-specification probability (ISP) to ascertain fitness-for-purpose of the proposed method. Microsoft Excel workbooks were developed for both TMU calculation approaches in addition to calculation of procedure capability index (Ppk) and sigma level for each analyte and attached in the Supplementary material. Moreover, linearity of the method underwent additional assessment using the previously mentioned TMU and procedure capability determination techniques. The robustness of the method was checked using Plackett-Burman experimental design (PBD) as an empirical modelling technique which was examined thoroughly to detect some interactions. The calibration graphs for each drug were rectilinear in the range of 0.34–7.08, 0.38–7.00 and 0.08–1.75 µg mL−1 for ADA, MPN, and PPN, respectively. The stress testing revealed photostability of the three components and variable degrees of degradation in the other conditions. The proposed HPLC-DAD method was successfully validated and applied for the determination of the investigated analytes in a gel matrix. In addition, method blueness and greenness were comparatively evaluated via very recent approaches of Blue Applicability Grade Index (BAGI), Green Solvents Selection Tool (GSST), Spider Diagram for Assessment of the Green Analytical Index (SDAGI), Analytical Method Greenness Score (AMGS), Carbon Footprint (CF), Modified Complementary Green Analytical Procedure Index (ComplexMoGAPI), and Analytical GREEnness Metric Approach (AGREE). Furthermore, the method whiteness was assessed via Red Green Blue Twelve (RGB 12) model following a relative ranking approach.

Development of a Reversed-Phase UPLC Method for Assay of Fipronil Including Determination of Its Related Substances in Bulk Batches of Fipronil Drug Substance.

Fipronil is a commonly used pesticide in the agricultural and animal health industries for the protection of crops and control of fleas, ticks, and chewing lice. It is difficult to obtain reproducible retention time and relative retention time (RRT) for a common hydrolytic degradation product of fipronil with the current European Pharmacopeia (EP) monograph for assay and estimation of related substances of fipronil. This situation causes misidentification, mislabeling, and/or false out-of-specification results for this hydrolytic degradation product of fipronil in bulk commercial batches during batch release and/or in the stability samples during the shelf life of a released batch. This study aimed to develop a reversed-phase ultra performance liquid chromatography (UPLC) method for assay and identification of fipronil including identification and estimation of its related substances in bulk drug substance batches of fipronil and provide consistent retention time of the hydrolytic degradation product. Fipronil and its related substances were separated by gradient elution on a Halo C18 column (50 mm × 2.1 mm id, 2.0 µm particle size) maintained at 40°C with 0.1% H3PO4 in H2O as mobile phase-A and acetonitrile-methanol (50 + 50, v/v) as mobile phase-B. Fipronil and its related substances were detected and quantified at 280 nm with a quantitation limit of 0.05% of the target (analytical) concentration. The UPLC method was able to separate all analytes of interest by gradient elution with a total run time of 7 min (approximately 40% faster than EP). In this paper, we report the development and validation of a fast, stability-indicating reversed-phase UPLC method for assay and estimation of related substances of fipronil in stability samples and bulk batches of fipronil. The new UPLC method is approximately 40% faster than the current Ph. Eur. monograph for fipronil assay and the new method provides reproducible retention of a common hydrolytic degradation product of fipronil.

Effect of Cimetidine on Metformin Pharmacokinetics and Endogenous Metabolite Levels in Rats.

Tubular secretion is a primary mechanism along with glomerular filtration for renal elimination of drugs and toxicants into urine. Organic cation transporters (OCTs) and multidrug and toxic extrusion (MATE) transporters facilitate the active secretion of cationic substrates, including drugs such as metformin and endogenous cations. We hypothesized that administration of cimetidine, an Oct/Mate inhibitor, will result in increased plasma levels and decreased renal clearance of metformin and endogenous Oct/Mate substrates in rats. A paired rat pharmacokinetic study was carried out in which metformin (5 mg/kg, intravenous) was administered as an exogenous substrate of Oct/Mate transporters to six Sprague-Dawley rats with and without cimetidine (100 mg/kg, intraperitoneal). When co-administered with cimetidine, metformin area under the curve increased significantly by 3.2-fold, and its renal clearance reduced significantly by 73%. Untargeted metabolomics was performed to investigate the effect of cimetidine on endogenous metabolome in the blood and urine samples. Over 8,000 features (metabolites) were detected in the blood, which were shortlisted using optimized criteria, i.e., a significant increase (P value < 0.05) in metabolite peak intensity in the cimetidine-treated group, reproducible retention time, and quality of chromatogram peak. The metabolite hits were classified into three groups that can potentially distinguish inhibition of i) extra-renal uptake transport or catabolism, ii) renal Octs, and iii) renal efflux transporters or metabolite formation. The metabolomics approach identified novel putative endogenous substrates of cationic transporters that could be tested as potential biomarkers to predict Oct/Mate transporter mediated drug-drug interactions in the preclinical stages. SIGNIFICANCE STATEMENT: Endogenous substrates of renal transporters in animal models could be used as potential biomarkers to predict renal drug-drug interactions in early drug development. Here we demonstrated that cimetidine, an inhibitor of organic cation transporters (Oct/Mate), could alter the pharmacokinetics of metformin and endogenous cationic substrates in rats. Several putative endogenous metabolites of Oct/Mate transporters were identified using metabolomics approach, which could be tested as potential transporter biomarkers to predict renal drug-drug interaction of Oct/Mate substrates.

Development of Liquid Chromatography-Mass Spectrometry (LC-MS) Method for Quantification of Broccoli Sulforaphane

Sulforaphane (SFN) is an isothiocyanate and the product of the hydrolysis of glucoraphanin (GRA) by myrosinase. Broccoli is one of the rich sources of GRA and thus SFN. SFN possess a wide range of bioactivities and is considered an anti-cancer phytochemical. Most of the current common methods used to quantify SFN are based on high-pressure liquid chromatography (HPLC) with diode array detection (DAD) — also known as HPLC-DAD. Although this technique has shown encouraging results, the detection of SFN by DAD is relatively weak and affected by high interference of sample matrices. Therefore, the aim of this study was to develop a liquid chromatography-mass spectrometry (LC-MS) method in which SFN is identified by molecular mass to give more accurate results. The developed method demonstrated a highly reproducible retention time (7.204 ± 0.008 min), producing a sharp, symmetrical and well-defined sulforaphane peak in standard and test samples. The most dominant ion of sulforaphane in the pure and test samples was 178 m/z ([M + H]+). In terms of linearity, the calibration curve had a coefficient of determination (R2) of 0.9963. The limit of detection of this method is 1.3 ng/mL, and the limit of quantification is 3.9 ng/mL, indicating high sensitivity. The uniformity of peak shape and retention time in both pure and test samples were the same suggesting excellent selectivity. Overall, the developed method showed promising results in identifying and quantifying broccoli SFN.

The Significance of System Suitability in High-Performance Liquid Chromatography (HPLC) Analysis: Ensuring Accurate and Reliable Results

High-Performance Liquid Chromatography (HPLC) has emerged as a fundamental technique in modern analytical chemistry, serving as a powerful tool for the separation, identification, and quantification of [1] diverse compounds. One critical aspect that ensures the validity and credibility of HPLC results is the concept of system suitability. This abstract highlights the importance of system suitability in HPLC analysis, emphasizing its role in ensuring accurate and reliable results. System suitability is a set of pre-defined criteria that evaluate the performance of the HPLCsystem, encompassing factors such as resolution, peak symmetry, tailing factor, retention time, and peak area reproducibility. Adhering to these criteria is essential for consistent and precise analyses, as they confirm that the instrument is operating optimally and capable of generating dependable data

B-297 Effortless and Streamlined Workflow for Absolute Quantitation of Therapeutic Monoclonal Antibodies Using Promise Proteomics mAbXmise Kits and TSQ Altis Plus Mass Spectrometer

Abstract Background Laboratories continuously seek improved productivity and efficacy for clinical testing, ultimately impacting the turn-around time and sample throughput. Various efforts have been made including rapid and high-throughput testing, automation, and high-end instrumentation. This also applies to therapeutic drug monitoring (TDM) of therapeutic monoclonal antibodies (mAbs). In particular, mass spectrometry (MS) has gained significant popularity in clinical laboratories for TDM of mAbs due to its great versatility to measure such complex biological proteins qualitatively and quantitatively. Here we present the streamlined workflow for the absolute quantitation of therapeutic mAbs using the Promise Proteomics mAbXmise kits and the Thermo Scientific™ TSQ Altis™ Plus mass spectrometer. The mAbXmise kit is the first commercial ready-to-use kit that offers all the necessary reagents for the LC-MS/MS assay including the stable-isotope labeled (SIL) proteins of the target mAbs (SIL-mAbs). The TSQ Altis™ Plus mass spectrometer is the latest generation of the TSQ MS series, offering superior acquisition speeds, sensitivity, selectivity, and robustness for routine and high-volume targeted protein quantitation. Methods The experimental workflow followed the instructions provided by Promise Proteomics with minor modifications (refer to instructions manual—https://customer.mabxmise.com). The kits used in this study include pre-coated plates with a full-length SIL-mAb corresponding to each target mAb sequence as internal standards with purity &amp;gt;95% and isotopic corporation &amp;gt;98%. Thus, the ITDM1 kit contains 2 SIL-mAbs and the OTDM1 kit contains 7 SIL-mAbs pre-coated in each plate. The kits also provide buffer stocks, reagents, 6 calibration standards including zero, and 2 QC controls. Each kit provides 12 strips capable of 96 sample preparation. The calibrators and QC samples were added to the mAbXmise plate and subjected to immunocapture followed by trypsin digestion. LC-MS/MS was performed using a Thermo Scientific™ Vanquish™ Flex UHPLC system hyphenated to TSQ Altis™ Plus mass spectrometer. Results Highly comparable results were observed across six replicates for both OTDM1 and ITDM1 kits with R2 values &amp;gt;0.99 and % RSD of peak areas of peptides &amp;lt;15. After confirming the complete sample preparation of 96 samples, an evaluation of LC-MS analytical performance was performed. The results show great linearity with R2 &amp;gt; 0.998 over measured calibration concentration points for all the OTDM1 and ITDM1 target peptides, respectively. Also, the data support the reproducible LC-MS measurement of the entire plate providing % RSD &amp;lt;10. The accuracy of QC samples was determined to be between 94 and 103% for OTDM1 peptides with a precision of &amp;lt;7% CV and between 95 and 107 % for ITDM peptides with a precision of &amp;lt;9% CV. The response of CAL1 (LLOQ) was 5 times higher than CAL0 for all the target peptides with accuracy between 96 and 112% and precision of &amp;lt;11% CV. Additionally, extremely reproducible retention times were observed from all SIL-mAb peptides with RT difference ±0.03 min during the evaluation. Conclusion The workflow tested in this report generated sensitive and robust data with high confidence, emphasizing the capability of MS-based TDM of mAbs as routine testing.

The differentiation of N-butyl pentylone isomers using GC-EI-MS and NMR

Forensic laboratories are faced with an ever-expanding seized drug landscape including the increasing prevalence of novel psychoactive substances (NPS), such as synthetic cathinones, that have varying potencies and scheduling. This study demonstrates a combined gas chromatography-electron ionization-mass spectrometry (GC-EI-MS) and nuclear magnetic resonance (NMR) spectroscopy approach for the differentiation of N-butyl pentylone isomers based on distinct retention times, characteristic EI mass spectra, and NMR characterization. Retention time reproducibility was assessed from 60 replicate measurements for each isomer over the course of a month. In addition, the effect of the mass spectrometer tune and the stability of an identified characteristic ion ratio using spectral data from ± 1 scan on either side of the peak apex were also statistically assessed using Welch’s ANOVA testing.The presence of diastereomers for N-sec-butyl pentylone was identified using the developed GC-EI-MS method, which was confirmed using one-dimensional and two-dimensional NMR spectroscopy. The retention time reproducibility of the chromatographic method was ± 0.076% or less over the course of a month. An identified characteristic ion ratio between the abundance of the fragment ion at m/z 128 and the fragment ion at m/z 72 enabled the differentiation of the four N-butyl pentylone isomers, even when accounting for the effect of the mass spectrometer tune and mass spectral scans used to calculate the characteristic ion ratio. The 95% confidence interval mean abundance ratio of the fragment ions at m/z 128 and m/z 72 was 17.14 ± 0.14 for N-butyl pentylone, 6.44 ± 0.05 for N-isobutyl pentylone, 3.38 ± 0.02 for N-sec-butyl pentylone, and 0.75 ± 0.01 for N-tert-butyl pentylone. These results highlight the capabilities of a combined GC-EI-MS and NMR approach for the differentiation and characterization of synthetic cathinone isomers.

Vacuum Insulated Probe Heated Electrospray Ionization Source Enhances Microflow Rate Chromatography Signals in the Bruker timsTOF Mass Spectrometer.

By far the largest contribution to ion detectability in liquid chromatography-driven mass spectrometry-based proteomics is the efficient generation of peptide molecular ions by the electrospray source. To maximize the transfer of peptides from the liquid to gaseous phase and allow molecular ions to enter the mass spectrometer at microspray flow rates, an efficient electrospray process is required. Here we describe the superior performance of newly design vacuum insulated probe heated electrospray ionization (VIP-HESI) source coupled to a Bruker timsTOF PRO mass spectrometer operated in microspray mode. VIP-HESI significantly improves chromatography signals in comparison to electrospray ionization (ESI) and nanospray ionization using the captivespray (CS) source and provides increased protein detection with higher quantitative precision, enhancing reproducibility of sample injection amounts. Protein quantitation of human K562 lymphoblast samples displayed excellent chromatographic retention time reproducibility (<10% coefficient of variation (CV)) with no signal degradation over extended periods of time, and a mouse plasma proteome analysis identified 12% more plasma protein groups allowing large-scale analysis to proceed with confidence (1,267 proteins at 0.4% CV). We show that the Slice-PASEF VIP-HESI mode is sensitive in identifying low amounts of peptide without losing quantitative precision. We demonstrate that VIP-HESI coupled with microflow rate chromatography achieves a higher depth of coverage and run-to-run reproducibility for a broad range of proteomic applications. Data and spectral libraries are available via ProteomeXchange (PXD040497).

Advanced Liquid Chromatography with Tandem Mass Spectrometry Method for Quantifying Glyphosate, Glufosinate, and Aminomethylphosphonic Acid Using Pre-Column Derivatization.

Analytical limitations make it challenging to develop effective methodologies for understanding glyphosate-based herbicide levels in drinking water and groundwater. Due to their lack of chromophores and zwitterionic nature, glyphosate-based herbicides are difficult to detect using traditional methods. This paper offers a straightforward method for quantifying glyphosate, glufosinate, and aminomethylphosphonic acid (AMPA) via 9-fluorenylmethylchloroformate (FMOC-Cl) pre-column derivatization and analysis by liquid chromatography with tandem mass spectrometry (LC-MS/MS). Method development was focused on optimizing the critical variables for optimal derivatization using a 24-factorial design. We found that complete derivatization significantly depends on the inclusion of borate buffer to create the alkaline conditions necessary for aminolysis. Ethylenediaminetetraacetic acid (EDTA) addition was critical to minimize metallic chelation and ensure reproducible retention times and peaks. However, EDTA concentrations ≥5% decreased peak intensity due to ion suppression. The FMOC-Cl concentration and derivatization time exhibited a direct proportional relationship, with the complete reaction achieved with 2.5 mM FMOC-Cl after 4 h. Concentrations of FMOC-Cl greater than 2.5 mM led to the formation of oxides, which interfere with the detection sensitivity and selectivity. Desirable results were achieved with 1% EDTA, 5% borate, and 2.5 mM FMOC-Cl, which led to complete derivatization after 4 h.

Development and validation of a rapid GC–MS method for seized drug screening

Controlled substances are one of the most commonly analyzed types of evidence in forensic laboratories. Persistent, high levels of case submissions in recent years have created a need for rapid screening techniques to decrease case backlogs and expedite confirmatory analyses. The most utilized technique for the analysis of seized drugs is gas chromatography mass spectrometry (GC-MS). While this approach typically takes tens of minutes, advances in the technique have enabled approximately 1 min run times with a short column and a rapid (10’s °C/s) temperature program. These advances were used in this work to develop and validate a rapid GC-MS method for screening seized drug samples. Method validation was completed to determine retention time repeatability and reproducibility, limits of detection, identification accuracy, selectivity, and carryover. Validation results showed the utility of this method for accurate and precise qualitative analyses for the screening of seized drugs across seven different drug classes. The optimized method was then applied to 15 real and mock case samples from the Maryland State Police Forensic Sciences Division. Controlled substances, cutting agents, and other diluents were identified from these samples using acceptance criteria of retention time and mass spectral comparisons currently used in casework. Using rapid GC-MS methods in casework could enable faster and more reliable presumptive testing results for the analysis of seized drug exhibits.

Simultaneous determination of heparan sulfate, chondroitin/dermatan sulfates, and hyaluronan glycosaminoglycan disaccharides by high-performance liquid chromatography using a reverse-phase column with adamantyl groups

Glycosaminoglycans (GAGs), which are one of the major components of proteoglycans, play a pivotal role in physiological processes such as signal transduction, cell adhesion, growth, and differentiation. Characterization of GAGs is challenging due to the tremendous structural diversity of heteropolysaccharides with numerous sulfate or carboxyl groups. In this present study, we examined the analysis of 2-aminobenzamide (2-AB) labeled GAG disaccharides by high-performance liquid chromatography (HPLC) using a reverse-phase (RP)-column with adamantyl groups. Under the analytical conditions, 17 types of 2-AB labeled GAG disaccharides derived from heparan sulfate, chondroitin/dermatan sulfates, and hyaluronan were sequentially separated in a single analysis. The analysis time was fast with high retention time reproducibility. Moreover, the RP-HPLC column with adamantyl groups allowed the quantification of GAGs in various biological samples, such as serum, cultured cells, and culture medium.

Robust Capillary- and Micro-Flow Liquid Chromatography-Tandem Mass Spectrometry Methods for High-Throughput Proteome Profiling.

Capillary- and micro-flow liquid chromatography-tandem mass spectrometry (capLC-MS/MS and μLC-MS/MS) is becoming a valuable alternative to nano-flow LC-MS/MS due to its high robustness and throughput. The systematic comparison of capLC-MS/MS and μLC-MS/MS systems for global proteome profiling has not been reported yet. Here, the capLC-MS/MS (150 μm i.d. column, 1 μL/min) and μLC-MS/MS (1 mm i.d. column, 50 μL/min) systems were both established based on UltiMate 3000 RSLCnano coupled to an Orbitrap Exploris 240 by integrating with different flowmeters. We evaluated both systems in terms of sensitivity, analysis throughput, separation efficiency, and robustness. capLC-MS/MS was about 10 times more sensitive than μLC-MS/MS at different gradient lengths. Compared with capLC-MS/MS, μLC-MS/MS was able to achieve higher analysis throughput and separation efficiency. During the 7 days' long-term performance test, both systems showed good reproducibility of chromatographic full width (RSD &lt; 3%), retention time (RSD &lt; 0.4%), and protein identification (RSD &lt; 3%). These results demonstrate that capLC-MS/MS is more suitable for high-throughput analysis of clinical samples with a limited starting material. When enough samples are available, μLC-MS/MS is preferred. Together, capLC and μLC coupled to Orbitrap Exploris 240 with moderate sensitivity should well meet the needs of large-cohort clinical proteomic analysis.

Ultrahigh-performance supercritical fluid chromatography for intraclass separation of lipids: Investigation of general principles

Powerful chromatographic techniques are required for lipidomic analyses due to the extreme complexity of natural lipidomes. In the past few years, ultrahigh-performance supercritical fluid chromatography (UHPSFC) has proven to be a good alternative to conventional LC methods for comprehensive lipidomic analysis. The goal of this work was to study UHPSFC intraclass separation of lipids according to the fatty acyl composition. The effects of column chemistry, mobile phase composition and gradient, flow rate, back pressure, temperature, and column coupling on intraclass separation of lipids were widely investigated and discussed. In general, UHPSFC exhibited interclass selectivity together with intraclass separation of lipids according to their total number of double bonds and acyl chain lengths. Moreover, separations of diacylglycerol and lysophosphatidylcholines regioisomers were achieved in some cases. The nature of the stationary phase showed the most prominent effect on UHPSFC intraclass selectivity, while other chromatographic conditions were used for partial improvement in resolution of lipid species. An octadecyl-based stationary phase showed excellent separation of nonpolar lipid species, including triacylglycerol isobars; however, it provided poor peak shapes and limited retention time reproducibility for polar lipids. Diol- and 1-aminoanthracene-based columns provided the best inter- and intraclass resolution of most lipids. The main benefit for UHPSFC separation of complex lipid samples is the combination of the acyl chain/double bond intraclass separation of lipids with excellent lipid class selectivity, which can facilitate mass spectrometry detection and quantitation of trace species without ion suppression effects.

Characterization of Cabernet Sauvignon Wines by Untargeted HS-SPME GC-QTOF-MS.

Untargeted metabolomics approaches are emerging as powerful tools for the quality evaluation and authenticity of food and beverages and have been applied to wine science. However, most fail to report the method validation, quality assurance and/or quality control applied, as well as the assessment through the metabolomics-methodology pipeline. Knowledge of Mexican viticulture, enology and wine science remains scarce, thus untargeted metabolomics approaches arise as a suitable tool. The aim of this study is to validate an untargeted HS-SPME-GC-qTOF/MS method, with attention to data processing to characterize Cabernet Sauvignon wines from two vineyards and two vintages. Validation parameters for targeted methods are applied in conjunction with the development of a recursive analysis of data. The combination of some parameters for targeted studies (repeatability and reproducibility < 20% RSD; linearity > 0.99; retention-time reproducibility < 0.5% RSD; match-identification factor < 2.0% RSD) with recursive analysis of data (101 entities detected) warrants that both chromatographic and spectrometry-processing data were under control and provided high-quality results, which in turn differentiate wine samples according to site and vintage. It also shows potential biomarkers that can be identified. This is a step forward in the pursuit of Mexican wine characterization that could be used as an authentication tool.

MEMS GC Column Performance for Analyzing Organics and Biological Molecules for Future Landed Planetary Missions

We present a novel, innovative approach to gas chromatography-mass spectrometry (GC-MS) based on micro-electro-mechanical systems (MEMS) columns that improve the current, state-of-the-art by dramatically reducing the size, mass, and power resources for deploying GC for future landed missions. The outlet of the MEMS GC column was coupled to a prototype of the MAss Spectrometer for Planetary EXploration (MASPEX) through a heated transfer line into the ion source. MEMS GC-MS experiments were performed to demonstrate linearity of response and establish limit of detection (LOD) to alkanes (organics), fatty acid methyl esters (FAMEs) and chemically derivatized amino acids (biological molecules). Linearity of response to each chemical family was demonstrated over two orders of magnitude dynamic range and limit of detection (LOD) values were single to tens (4–43) of picomoles per 1 μl injection volume. MEMS GC column analytical performance was also demonstrated for a “Mega Mix” of chemical analytes including organics and biological molecules. Chromatographic resolution exceeded 200, retention time reproducibility was &amp;lt;&amp;lt; 1% RSD (majority ≤ 0.3%), and peak capacity values calculated to be 124 ± 2 over a 435 s retention time window. The 5.5 m MEMS column was also shown to be a suitable alternative to traditional commercial columns for use in comprehensive two-dimensional gas chromatography (GC × GC). Mass spectra collected from MASPEX showed close consistency with National Institute of Technology (NIST) reference mass spectra and were used for high confidence identification of all eluting analytes.

2-μm fused-core column ultra-high-performance liquid chromatography/tandem mass spectrometric determination of donepezil in human plasma: Application to a bioequivalence study

Ultra-high-performance liquid chromatography (UHPLC) hyphenated to tandem mass spectrometric methods using the dimethylpentafluorophenylpropyl stationary phase with polar and nonpolar characteristics of a 2-μm fused-core silica particle packing were evaluated to perform efficient separation for the analysis of donepezil in human plasma. The fused-core silica particle design providing the shorter diffusional mass transfer path for the analytes is less affected in resolving power by increasing mobile-phase velocity for faster chromatographic resolution between the administered compound and multiple interfering peaks from the extracted quality control, calibration standard and study samples following simple protein precipitation extraction procedures. In this work, two UHPLC-MS/MS approaches requiring 1.2 min per sample run time and neither expensive ultra-high-pressure instrumentation nor new laboratory protocols were applied and compared for determination of donepezil in human plasma at sub-nanograms per milliliter region to support a randomized, crossover bioequivalence (BE) study in which healthy volunteers each received a single oral dose of the test and reference formulations of 10 mg donepezil hydrochloride. The column performance in terms of chromatographic separation efficiency, peak asymmetry and resolution and retention time reproducibility was found to be sustainable over a thousand extracted plasma injections. The linear dynamic range was detected over a concentration range of 0.2–50 ng/mL. The intra- and inter-day assay accuracy and precision were within 15% for the analyte in individual biological fluids. Positive correlation coefficients (r) greater than 0.98 and 0.99 for donepezil concentrations in study plasma samplers measured by the proposed UHPLC-MS/MS approaches and another validated HPLC-MS/MS method were observed.

Ultrafast liquid chromatography-tandem mass spectrometry determination of donepezil in human plasma: application to a bioequivalence study.

A liquid chromatography equipped with tandem mass spectrometric method using multi-stage flow rates was developed for the determination of donepezil in human plasma to support a randomized, crossover bioequivalence (BE) study in which healthy volunteers each received a single oral dose of the reference and test formulations of 10 mg donepezil hydrochloride. This integrated liquid chromatography with tandem mass spectrometry (LC-MS/MS) system with electrospray ionization and a deuterium-labeled internal standard (IS) were employed for the positive multiple-reaction-monitoring (MRM) analyses. The baseline separation using a high-resolution monolithic column under gradient and flexible flowrate conditions between donepezil and multiple interfering peaks from the extracted quality control, calibration standard and study plasma samples following simple protein precipitation extraction procedures was accomplished within 1.5 minutes. The ultrafast monolithic column performance in terms of chromatographic separation efficiency, peak asymmetry and resolution and retention time reproducibility was found to be sustainable. The linear dynamic range was detected over a concentration range of 0.2–50 ng/mL. The intra- and inter-day assay accuracy and precision were within 15% for the analyte in individual biological fluids. A positive correlation coefficient (r) greater than 0.995 for donepezil concentrations in study plasma samplers measured by the proposed and the other validated LC-MS/MS methods in support of a bioequivalence study was observed.

Temperature responsive chromatography for therapeutic drug monitoring with an aqueous mobile phase

Therapeutic drug monitoring is a key technology for effective pharmacological treatment. In the present study, a temperature-responsive chromatography column was developed for safe and simple therapeutic drug monitoring without the use of organic solvents. Poly(N-isopropylacrylamide) (PNIPAAm) hydrogel-modified silica beads were prepared via a condensation reaction and radical polymerization. The temperature-dependent elution behavior of the drugs was observed using a PNIPAAm-modified silica-bead packed column and an all-aqueous mobile phase. Sharp peaks with reproducible retention times were observed at temperatures of 30 °C or 40 °C because the PNIPAAm hydrogel on the silica beads shrinks at these temperatures, limiting drug diffusion into the PNIPAAm hydrogel layer. The elution behavior of the sample from the prepared column was examined using a mixture of serum and model drugs. The serum and drugs were separated on the column at 30 °C or 40 °C, and the concentration of the eluted drug was obtained using the calibration curve. The results show that the prepared chromatography column would be useful for therapeutic drug monitoring because the drug concentration in serum can be measured without using organic solvents in the mobile phase and without any need for sample preparation.

S‐citalopram imprinted monolithic columns for capillary electrochromatography enantioseparations

In this study, the molecular imprinting method was used to separate enantiomeric forms of chiral antidepressant drug, R,S-citalopram (R,S-CIT) in aqueous solution by CEC system combining the advantages of capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC). For that, an amino acid-based molecularly imprinted monolithic capillary column was designed and used as a stationary phase for selective separation of S-citalopram (S-CIT) for the first time. S-CIT was selectively separated from the aqueous solution containing the other enantiomeric form of R-CIT, which is the same in size and shape as the template molecule. Morphology of the molecularly imprinted (MIP S-CIT) and non-imprinted (NIP S-CIT) monolithic capillary columns was observed by scanning electron microscopy. Imprinting efficiency of MIP S-CIT monolithic capillary column used for selective S-CIT separation was verified by comparing with NIP S-CIT and calculated imprinting factor (I.F:1.81) proved the high selectivity of the MIP S-CIT for S-CIT. Cavities formed for S-CIT form enabled selective (α = 2.08) separation of the target molecule from the other enantiomeric R-CIT form. Separation was achieved in a short period of 10 min, with the electrophoretic mobility of 7.68 × 10-6 m2 /Vs for R,S-CIT at pH 7.0 10mM PB and 50% ACN ratio. The performance of both MIP S-CIT and NIP S-CIT columns was estimated by repeating the R,S-CIT separations with intra-batch and inter-batch studies for reproducibility of retention times of R,S-CITs. Estimated RSD values that are lower than 2% suggest that the monolithic columns separate R,S-CIT enantiomers without losing separation efficiency.