LC-MS Method For Quantitation Research Articles

A quantitative LC–MS method to determine surface contamination of antineoplastic drugs by wipe sampling

The main objective was to develop a wipe sampling test to measure surface contamination of the most frequently used antineoplastic drugs (ADs) in Swedish healthcare and, furthermore, to develop an analysis method sensitive enough to assess low levels of contamination. Two wipe sampling tests with separate sample processing methods assessing (i) cyclophosphamide (CP), ifosfamide (IF), 5-fluorouracil (5-FU), etoposide (ETO), gemcitabine (GEM) and cytarabine (CYT) (Wipe Test 1); and (ii) GEM, CYT and methotrexate (MTX) (Wipe Test 2), respectively, were developed by optimization of absorption and extraction efficiencies using different wipe tissue materials, tissue wetting solution, and extraction solvents. A fast liquid chromatography tandem mass spectrometry method was developed for simultaneous detection of the studied ADs. The limit of quantification for the method was between 0.04 to 2.4 ng/wipe sample (0.10 to 6.1 pg/cm2 for an area of 400 cm2) and at 50 ng/sample the within-day precision was between 1.3 and 15%, and the accuracy between 102 and 127%. Wipe Test 1 was applied in an assessment of cleaning efficiency of five different cleaning solutions (formic acid, water, sodium hydroxide, ethanol, and sodium dodecyl sulfate (SDS) for removal of ADs from surfaces made of stainless steel or plastic. For CP, IF, 5-FU, GEM, and CYT 92% of the AD were removed regardless of surface and cleaning solution. In conclusion, a user-friendly assessment method to measure low levels of seven ADs in the work environment was developed and validated. Assessment of the decontamination efficiency of cleaning solutions concerning removal of ADs from stainless steel showed that efficiencies differed depending on the AD with water being the least effective cleaning agent. The results suggests that a combination of different cleaning agents including detergent and a solution with an organic component would be optimal to efficiently remove the measured ADs from surfaces in the workplace.

Per-pixel absolute quantitation for mass spectrometry imaging of endogenous lipidomes by model prediction of mass transfer kinetics in single-probe-based ambient liquid extraction

With the development of mass spectrometry imaging (MSI), techniques providing quantitative information on the spatial distribution have attracted more attentions recent years. However, for MSI of endogenous compounds in bio-samples, the uncertainty of locally varied sampling efficiencies always hinders accurate absolute quantitation. Here single-probe was used for ambient liquid extraction MSI in rat cerebellum, and standards of phosphatidylcholines (PCs) and cerebrosides (CBs) were doped in extraction solvent. The extraction kinetic curves of endogenous lipids in the ambient liquid extraction during probe parking in single pixel of tissue were investigated. From the results, the extraction kinetic curves were varied between different lipid species in different brain regions, resulting in variations of extraction efficiencies between imaging pixels, and calibration with standards deposited in tissue could not compensate for the variations. In our approach, the theoretical kinetic model of ambient liquid extraction was established, and original concentrations of endogenous lipids in each pixel of tissue were predicted by fitting the experimental extraction kinetic curve in each imaging pixel to the model. The experimental data was demonstrated to be well fitted to the kinetic model with R2 > 0.86, and only with 18-s extraction in each pixel, the original lipid concentrations were predicted accurately with relative errors <23%. With the new method, totally 157 lipids and small metabolites were imaged, and per-pixel quantitation was achieved for 19 PCs and 4 CBs. Compared with conventional quantitative MSI (q-MSI) method, the new q-MSI method had better reproducibility and wider linear range, and produced better contrast in the quantitative images of lipids in brain tissue with less hot spots and noises. The absolute quantitation results by the new method were verified by quantitative LC–MS method with Pearson'r > 0.9 and the slope of the linear fitting line of the correlation plot near 1.

Determination of Casein Allergens in Extensively Hydrolyzed Casein Infant Formula by Liquid Chromatography-Tandem Mass Spectrometry.

BackgroundThe use of hypoallergenic infant formulas and the need for reliable tests to determine the presence of residual antigens have increased in parallel.ObjectiveAn LC-MS method for quantitation of casein was validated using incurred samples and a matrix-matched external standard curve.MethodPowdered infant formula samples were extracted in a buffer of sodium deoxycholate and ammonium bicarbonate at 60°C and filtered through 7 kDa desalting columns. Samples were digested overnight with trypsin and precipitated with acid prior to analysis of marker peptides by tandem mass spectrometry.ResultsBased on three marker peptides, the linear range for casein was 1.8–42 μg/g of powdered infant formula with an LOQ of 1.8 μg/g. The determination coefficients (R2) for each curve were ≥0.99 for casein peptides. Method repeatability was ≤22% RSD and intermediate precision was ≤23% RSD; recovery of casein from incurred material (2–20 µg/g) ranged from 78% to 118%.ConclusionsAn LC-MS/MS method was developed and validated for confirmation of casein allergens in hypoallergenic infant formula.HighlightsA method was developed to accurately and reliably quantify casein allergens in extensively hydrolyzed casein infant formula by LC-MS without the need for custom peptide standards.

Development of a targeted nanoLC-MS/MS method for quantitation of residual toxins from Bordetella pertussis

Whooping cough is a highly contagious respiratory disease caused by Bordetella pertussis (B. pertussis) infection. Pertussis pathogenesis is driven by cell-surface adhesion proteins and secreted toxins; some of which have been harnessed for their immunogenic properties as purified antigen components in acellular vaccines. Two of these virulence factors, adenylate cyclase toxin (ACT) and dermonecrotic toxin (DNT), are protein toxins with potential for co-purification, and therefore must be monitored as process-related impurities during the development of acellular Pertussis vaccine candidates. Here we describe the development of a targeted nanoLC-MS/MS method for absolute quantitation of ACT and DNT in process intermediates from acellular Pertussis antigen purification. Starting from an in silico digest of the toxin sequences, a synthetic peptide screening approach was applied to systematically evaluate candidate sequences as surrogates for protein quantitation. Following refinement to a subset of sequences, absolutely quantified heavy-labelled (AQUA) peptides were implemented in a parallel reaction monitoring (PRM) workflow with limits of detection (LOD) and quantitation (LOQ) in the 12.5–25 amol (2−4 ng/mL) range on-column. In this work, we highlight a ‘standards-driven’ approach to surrogate peptide selection for protein quantitation. This strategy can be broadly applied in the absence of purified reference material and accelerate quantitative LC–MS method development across multiple sample matrices.

Development and fit-for-purpose verification of an LC-MS method for quantitation of hemagglutinin and neuraminidase proteins in influenza virus-like particle vaccine candidates

Recombinant influenza Virus-Like Particle (VLP) vaccines are promising vaccine candidates to prevent influenza, contain two major viral antigenic glycoproteins, Hemagglutinin (HA) and Neuraminidase (NA), on the surface of recombinant VLPs. Accurate quantitation of the mass of these antigenic proteins is important to ensure the product quality and proper dosing. Currently, Single Radial Immunodiffusion (SRID) is a recognized assay for determination of the HA immuno-reactive concentration (potency) in vaccine products, based on immuno-reactivity of HA with strain-specific antisera. The SRID assay, however, requires availability of strain-specific and properly calibrated reagents, which can be time-consuming to generate and calibrate. In addition, the assay is not suitable for quantitation of low abundant proteins, such as NA. In order to accelerate the overall production cycle, we have developed and optimized a high-resolution (HR) LC-MS method for absolute quantitation of both HA and NA protein concentrations in influenza VLP vaccine candidates. In this work, we present the method development, optimization and verification of its suitability for the intended purpose, as a prerequisite for its potential application in Quality Control, by assessing specificity, precision and accuracy, detection characteristics, and dynamic linear range. The method can be also used for other HA/NA containing preparations including in-process samples, purified proteins, whole virus preparations, nano-particle and egg-based vaccine preparations, or for calibration of SRID reference antigens.

A Simple LC-MS Method for the Quantitation of Alkaloids in Endophyte-Infected Perennial Ryegrass.

The rapid identification and quantitation of alkaloids produced by Epichloë endophyte-infected pasture grass is important for the agricultural industry. Beneficial alkaloids, such as peramine, provide the grass with enhanced insect protection. Conversely, ergovaline and lolitrem B can negatively impact livestock. Currently, a single validated method to measure these combined alkaloids in planta does not exist. Here, a simple two-step extraction method was developed for Epichloë-infected perennial ryegrass (Lolium perenne L.). Peramine, ergovaline and lolitrem B were quantified using liquid chromatography–mass spectrometry (LC–MS). Alkaloid linearity, limit of detection (LOD), limit of quantitation (LOQ), accuracy, precision, selectivity, recovery, matrix effect and robustness were all established. The validated method was applied to eight different ryegrass-endophyte symbiota. Robustness was established by comparing quantitation results across two additional instruments; a triple quadruple mass spectrometer (QQQ MS) and by fluorescence detection (FLD). Quantitation results were similar across all three instruments, indicating good reproducibility. LOQ values ranged from 0.8 ng/mL to 6 ng/mL, approximately one hundred times lower than those established by previous work using FLD (for ergovaline and lolitrem B), and LC–MS (for peramine). This work provides the first highly sensitive quantitative LC–MS method for the accurate and reproducible quantitation of important endophyte-derived alkaloids.

Simultaneous quantitative LC–MS method of ketamine, midazolam and their metabolites (dehydronorketamine, norketamine and 1hydroxymidazolam) for its application in patients on extracorporeal membrane oxygenation (ECMO) therapy

Ketamine (Ket) and midazolam (MDZ) are commonly administered drugs in the intensive care setting for analgesia and sedation. Ket and MDZ are metabolized to dehydro-norketamine (DHNK), nor-ketamine (NK) and 1-hydroxy midazolam (1HMDZ). Limited studies evaluating their pharmacokinetics exists in patients on extracorporeal membrane oxygenation (ECMO) therapy. Therefore, we developed a quantitative, high-performance liquid chromatography-mass spectrometry (with single ion monitoring) method to simultaneously detect Ket, MDZ and their (DHNK, NK and 1HMDZ) metabolites in human plasma. Considerable sensitivity was obtained for the analytes using a C18 HILIC column operated by a high-performance liquid chromatography system coupled with a Thermo Exactive Orbitrap mass spectrometer. Calibration curves were developed for analyte molecules (n = 5) in the presence of carbamazepine (CBZ) as an internal standard. The lower limits of quantitation (LLOQ) for Ket and MDZ were 20 and 10 ng/mL, respectively with the LLOQ for DHNK, NK and 1HMDZ at 470, 320 and 150 ng/ml. Moreover, the percent coefficient of variance and precision for inter- and intra-day runs were within the standards set forth by the ICH and FDA guidelines. This method is sensitive and has been successfully applied to an ongoing pharmacokinetic study in patients on ECMO therapy.

Energy resolved mass spectrometry of chlorogenic acids and its application to isomer quantification by direct infusion tandem mass spectrometry.

With the advent of high-perfomance liquid chromatography (HPLC)-tandem mass spectrometry (MS) using ion trap mass analysers it is possible to acquire unambigious structural information in particular with respect to aspects of regiochemistry and stereochemistry of organic compounds present in complex mixtures such as coffee extracts. However, HPLC-MS methods are resource extensive, laborious and lacking user friendliness. To introduce a simple parameter - the energy threshhold for fragmentation - determined using energy resolved MS and demonstrate its value for the complete structural characterisation and even relative quantification of individual isomeric chlrogenic acids in direct infusion experiments. Monocaffeoyl and dicaffeoyl quinic acids were investigated by direct infusion energy resolved mass spectrometry (ER-MS) in negative in mode, using a quadrupole ion trap and quadrupole time-of-flight (Q-TOF) mass spectrometer. Methanolic coffee extracts were quantitatively investigated by HPLC-MS and direct infusion ER-MS. Fragmentation occurs with retention of regiochemistry and regiochemistry of fragment ions can be determined using ER-MS. Analysis of breakdown graphs allows extraction of a single numerical parameter that allows assignment of regiochemistry. Analysis of monocaffeoyl and dicaffeoyl quinic acids revealed that regiosiomers could be distinguished and assigned based on their dissociation energies in collisional induced activation. Furthermore relative quantification of regioisomers by direct infusion ER-MS is possible within an error range of ±10% if compared with a conventional quantitative LC-MS method. ER-MS can be exploited in determining relative isomers quantities of chlorogenic acids (CGAs) in crude plant extracts by direct infusion tandem MS omitting time and resource intensive chromatographic separation.

CLSI C62-A: A New Standard for Clinical Mass Spectrometry.

Until recently, there existed minimal guidance on the use of LC-MS for clinical diagnostics. The Clinical and Laboratory Standards Institute (CLSI)2 has now presented a standardized approach for LC-MS assay development and verification in its new guidance document, CLSI C62-A. This document should aid in the harmonization of LC-MS methods and thus have significant ramifications for the evolution of this technology in the clinical laboratory. The ability to accurately identify and quantify a measurand with high sensitivity and specificity by use of selective reaction monitoring has been the driving force for the adoption of LC-MS in the clinical laboratory. LC-MS offers analytical specificity superior to that of widespread diagnostic methodologies such as immunoassays and enzymatic assays. Whereas immunoassays are commonplace in the clinical laboratory, they are not available for all analytes of interest, are plagued by specificity issues, and require the time-consuming production and evaluation of antibodies during development. Just as immunoassays have evolved since their development >50 years ago and have found their place as the leading automated technological approach to diagnostic testing, mass spectrometry (MS) will continue to progress and influence the clinical laboratory landscape. Therefore, standardization of the use of LC-MS is essential. Mass spectrometers were initially used in clinical laboratories only for specialized testing by highly trained and experienced operators. Today they are essential for the diagnosis of metabolic disorders, screening of diseases, quantification of hormone concentrations, monitoring of drug therapies, identification of microbial organisms, and recognition of drug toxicity and poisonings. Without the current clinical quantitative LC-MS methods, metabolic disorders would go undetected at birth, the accuracy of testosterone measurements for women and children would be equivalent to an educated guess, and monitoring of a variety of chemotherapeutics and immunosuppressants would be limited. Despite its widespread use, there is only 1 quantitative LC-MS assay …

Quantitative LC–MS determination of liposomal encapsulated prednisolone phosphate and non-encapsulated prednisolone concentrations in murine whole blood and liver tissue

The underlying pharmacokinetic profile of liposomal drug delivery systems is not yet fully known. This is primarily due to a lack of suitable quantitative bioanalytical methodology to simultaneously determine separate liposomal-encapsulated and non-encapsulated drug tissue concentrations in complex biological samples. Here, an LC–MS method was developed which enables the simultaneous quantification of separate liposomal-encapsulated prednisolone phosphate and non-encapsulated prednisolone concentrations in whole blood and liver tissue.Liquid chromatography, negative electrospray ionization and Orbitrap-MS analysis allowed highly accurate and sensitive detection of prednisolone phosphate (PP) and prednisolone (P) in complex matrix. Using dexamethasone phosphate and dexamethasone as internal standards, the quantitative LC–MS method was optimized and validated for high selectivity, sensitivity and quantitative accuracy of PP and P from liposomes.The lower limits of quantitation were 0.99μmol/L blood and 0.53nmol/g liver for PP, and 229nmol/L blood and 0.514nmol/g liver for P. Quantitative accuracies of 84–118% were observed. The intra-run precision was ≤11%.Application of this new LC–MS method will yield the first liposomal pharmacokinetic profile showing accurate encapsulated and non-encapsulated drug tissue concentrations separately. This is also the first quantitative LC–MS method for the simultaneous quantification of the prodrug PP and its parent drug P in whole blood and liver tissue samples.

Clinical Utility of Insulin-Like Growth Factor 1 and 2; Determination by High Resolution Mass Spectrometry

Measurement of insulin-like growth factor-1 (IGF-I) has utility for the diagnosis and management of growth disorders, but inter-assay comparison of results has been complicated by a multitude of reference standards, antibodies, detection methods, and pre-analytical preparation strategies. We developed a quantitative LC-MS method for intact IGF-I, which has advantages in throughput and complexity when compared to mass spectrometric approaches that rely on stable isotope dilution analysis of tryptic peptides. Since the method makes use of full-scan data, the assay was easily extended to provide quantitative measurement of IGF-II using the same assay protocol. The validated LC-MS assay for IGF-I and IGF-II provides accurate results across the pediatric and adult reference range and is suitable for clinical use.

A One-Step Solid Phase Extraction Method for Bioanalysis of a Phosphorothioate Oligonucleotide and Its 3′ n-1 Metabolite from Rat Plasma by uHPLC–MS/MS

Oligonucleotide therapeutics have emerged as a promising class of drugs to treat a wide range of diseases caused by genetic abnormalities. Replacement of the phosphodiester linkage with a phosphorothioate is one of the most successful modifications made to oligonucleotides to enhance their in vivo stability. The longer elimination phase of phosphorothioates and other modified oligonucleotides requires sensitive and selective methods to quantify the parent drug and their metabolites simultaneously. Liquid chromatography tandem mass spectrometry has excellent selectivity between the parent drug and its metabolites and a wide dynamic range. However, the biological sample extraction remains a formidable challenge in developing quantitative LC-MS methods for oligonucleotides. Protein precipitation, protein digestion, liquid-liquid extraction, reversed phase solid phase extraction (SPE), strong anion exchange SPE, and combinations of them have been reported to extract oligonucleotides from biological matrices. Unfortunately, these methods either have low recoveries or present potential problems for applications with chromatography due to the large amount of matrix substances in the resulting solutions. In this study, a weak anion exchange SPE method was optimized. The recovery ranged from 60% to 80% depending on the concentration. This is the first report of a one-step SPE method with recoveries greater than 60% across the method dynamic range. This sample extraction procedure was used in combination with ultrahigh-performance liquid chromatography-tandem mass spectrometry. The lower limit of quantitation was 10ng/mL (1.3nM), and the dynamic range was 10-1,000ng/mL. The intra- and inter-day precision and accuracy were within 8.4% and 10.5%, respectively.

Quantitative method development for measurement of Maesa lanceolata saponins by LC-MS

Triterpene saponins are a class of plant natural products with a wide range of bioactivities, which make them an interesting research subject. The small tree Maesa lanceolata, growing in African countries, is used in traditional medicine against various diseases. In previous work a triterpenoid saponin mixture was isolated from the leaves of Maesa lanceolata and the compounds were identified [1, 2]. The compounds showed virucidal, haemolytic, molluscicidal and anti-angiogenic activity [3, 4]. Here we report the development of a quantitative LC-MS method to analyse saponin compounds in crude root and leaf extracts of Maesa plants. The crude extract is first purified on a C18ec SPE column. Then the compounds are separated on a reversed phase C18 column (Grace Vydac, 3.2mm x 250mm –300Å) with a H2O/ACN (0.06% formic acid) gradient before analysis with mass spectrometry (Bruker Daltonics Esquire 3000 plus). This method will be used to screen plants whether or not induced by methyl-jasmonate, or plants such as Medicago truncatula transformed with genes involved in saponin biosynthesis, for the presence of Maesa saponins.