Quantification of the lung cancer tumor marker CYFRA 21-1 using protein precipitation, immunoaffinity bottom-up LC-MS/MS.

OBJECTIVES: Numerous studies have proven the potential of cytokeratin 19 fragment 21-1 (CYFRA 21-1) detection in the (early) diagnosis and treatment monitoring of non-small cell lung cancer (NSCLC). Conventional immunoassays for CYFRA 21-1 quantification are however prone to interferences and lack diagnostic sensitivity and standardization. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is an emerging approach based on a different, often superior, detection principle, which may improve the clinical applicability of CYFRA 21-1 in cancer diagnostics. Therefore, we developed and validated a protein precipitation, immunoaffinity (IA) LC-MS/MS assay for quantitative analysis of serum CYFRA 21-1. METHODS: Selective sample preparation was performed using ammonium sulfate (AS) precipitation, IA purification, tryptic digestion and LC-MS/MS quantification using a signature peptide and isotopically labeled internal standard. The workflow was optimized and validated according to EMA guidelines and results were compared to a conventional immunoassay. RESULTS: Significant interference effects were seen during IA purification, which were sufficiently solved by performing AS precipitation prior to IA purification. A linear calibration curve was obtained in the range of 1.0-100 ng/mL (R2=0.98). Accuracy and precision were well within acceptance criteria. In sera of patients suspected of lung cancer, the method showed good correlation with the immunoassay. CONCLUSIONS: A robust AS precipitation-IA LC-MS/MS assay for the quantification of serum CYFRA 21-1 was developed. With this assay, the clinically added value of LC-MS/MS-based detection over immunoassays can be further explored.

Development and validation of a liquid chromatography-tandem mass spectrometry method for the quantification of the acetyl-coenzyme A competitive p300/CBP catalytic inhibitor A-485 in biological samples.

The small molecule A-485 competitively inhibits the histone acetyltransferase domain of CBP (cyclic-adenosine monophosphate response element-binding protein)/p300. Apart from its antineoplastic activity, researchers are exploring its potential benefits in treating osteoporosis and its impact on energy metabolism. However, so far, only limited pharmacokinetic data are available, and the crucial determination of A-485 concentration in various biological materials with small sample volumes remains unpublished. A rapid and sensitive LC-tandem mass spectrometry method has been developed and validated to quantify A-485 in mouse serum and tissue. In this method, serum samples underwent precipitation with acetonitrile, while cell lysates were appropriately diluted. The determination of A-485 utilized a reversed-phase column with a mobile phase gradient, and detection was carried out in multiple reaction monitoring mode. The lower standard sample, with a concentration of 7.8 ng/mL, served as the lower limit of quantification, while the upper standard was established at 1000 ng/mL. A-485 concentrations were assessed in both serum samples and the lysate of all examined tissues, revealing swift metabolic clearance. The analytical method outlined here is deemed appropriate for subsequent studies. The ability to measure the active ingredient in various compartments facilitates the determination of accurate pharmacokinetic parameters. In the event of human use of A-485, the analysis method can be seamlessly transferred to human samples.

A Simple, Fast, Sensitive LC-MS/MS Method to Quantify NAD(H) in Biological Samples: Plasma NAD(H) Measurement to Monitor Brain Pathophysiology.

Nicotinamide adenine dinucleotide (NAD) is a cofactor in redox reactions and an essential mediator of energy metabolism. The redox balance between NAD+ and NADH affects various diseases, cell differentiation, and aging, and in recent years there has been a growing need for measurement techniques with improved accuracy. However, NAD(H) measurements, representing both NAD+ and NADH, have been limited by the compound's properties. We achieved highly sensitive simultaneous measurement of NAD+ and NADH under non-ion pairing, mobile phase conditions of water, or methanol containing 5 mM ammonium acetate. These were achieved using a simple pre-treatment and 7-min analysis time. Use of the stable isotope 13C5-NAD+ as an internal standard enabled validation close to BMV criteria and demonstrated the robustness of NAD(H) determination. Measurements using this method showed that brain NAD(H) levels correlate strongly with plasma NAD(H) levels in the same mouse, indicating that NAD(H) concentrations in brain tissue are reflected in plasma. As NAD(H) is involved in various neurodegenerative diseases and cerebral ischemia, as well as brain diseases such as mitochondrial myopathies, monitoring changes in NADH levels in plasma after drug administration will be useful for development of future diagnostics and therapeutics.

Predicting Colloidal Stability of High-Concentration Monoclonal Antibody Formulations in Common Pharmaceutical Buffers Using Improved Polyethylene Glycol Induced Protein Precipitation Assay.

Colloidal stability is an important consideration when developing high concentration mAb formulations. PEG-induced protein precipitation is a commonly used assay to assess the colloidal stability of protein solutions. However, the practical usefulness and the current theoretical model for this assay have yet to be verified over a large formulation space across multiple mAbs and mAb-based modalities. In the present study, we used PEG-induced protein precipitation assays to evaluate colloidal stability of 3 mAbs in 24 common formulation buffers at 20 and 5 °C. These prediction assays were conducted at low protein concentration (1 mg/mL). We also directly characterized high concentration (100 mg/mL) formulations for cold-induced phase separation, turbidity, and concentratibility by ultrafiltration. This systematic study allowed analysis of the correlation between the results of low concentration assays and the high concentration attributes. The key findings of this study include the following: (1) verification of the usefulness of three different parameters (Cmid, µB, and Tcloud) from PEG-induced protein precipitation assays for ranking colloidal stability of high concentration mAb formulations; (2) a new method to implement PEG-induced protein precipitation assay suitable for high throughput screening with low sample consumption; (3) improvement in the theoretical model for calculating robust thermodynamic parameters of colloidal stability (µB and εB) that are independent of specific experimental settings; (4) systematic evaluation of the effects of pH and buffer salts on colloidal stability of mAbs in common formulation buffers. These findings provide improved theoretical and practical tools for assessing the colloidal stability of mAbs and mAb-based modalities during formulation development.

Cation affinity purification of histidine-tagged proteins.

Protein purification is a basic technology in both biological research and industrial production, and efficient, convenient, economical, and environmentally friendly purification methods have always been pursued. In this study, it was found that alkaline earth metal cations (Mg2+, Ca2+) and alkali metal cations (Li+, Na+, K+) and even nonmetal cations (e.g., NH4+, imidazole, guanidine, arginine, lysine) can precipitate multi-histidine-tagged proteins (at least two tags in a whole protein) at low salts concentrations that are 1-3 orders of magnitude lower than salting-out, and precipitated proteins could be dissolved at moderate concentration of corresponding cation. Based on this finding, a novel cation affinity purification method was developed, which requires only three centrifugal separations to obtain highly purified protein with purification fold similar to that of immobilized metal affinity chromatography. The study also provides a possible explanation for unexpected protein precipitation and reminds researchers to consider the influence of cations on the experimental results. The interaction between histidine-tagged proteins and cations may also have broad application prospects. KEY POINTS: • Histidine-tagged proteins can be precipitated by low-concentrations common cations • A novel nonchromatographic protein purification method was developed • Purified protein can be obtained in pellet form by only three centrifugations.

Dispersive solid-phase extraction of risperidone from plasma samples using graphene oxide aerogels and determination with liquid chromatography.

A graphene oxide-based aerogel was synthesized and applied to the extraction and the determinations with the high-performance liquid chromatography-ultraviolet detector. After the characterization of the produced graphene-aerogel, it was utilized as a dispersive solid-phase extraction sorbent for risperidone extraction from plasma samples. Aerogels are materials with a large surface area-to-mass ratio and plenty of core with functional groups which can easily attach to the analytes to extract them to the second phase. The suggested method determined risperidone in plasma samples in the wide dynamic range from 20 ng/ml to 3 µg/ml. The limits of detection and quantification of the developed method were calculated as 2.4 and 8.2 ng/ml, respectively. As a novel feature, the developed method has no need to precipitate plasma proteins, improving the analytical performance of the analysis. Also, for the first time, the produced materials were utilized for the extraction of risperidone from the plasma samples. The obtained results revealed that the developed approach could be employed as an accurate method for the quantification of risperidone in real plasma samples.

Ultra-performance liquid chromatography-tandem mass spectrometry determination and pharmacokinetic studies of five phenolic acids in rat plasma after intravenous administration of salvianolic acid for injection.

A simple, efficient, and sensitive ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for simultaneous determination and pharmacokinetic study of salvianolic acid D, rosmarinic acid, lithospermicic acid, salvianolic acid B, and salvianolic acid Y in rat plasma after intravenous administration of salvianolic acid for injection. Three doses of administration, containing 10, 25, and 62.5 mg/kg, were investigated. Plasma samples were pretreated using protein precipitation with pre-cooled acetonitrile. As shown in S1, Chromatographic separation was achieved on a Waters Acquity UPLC® BEH C18 column (1.7 µm, 2.1 × 100 mm) with a mobile phase composed of acetonitrile-methanol-0.5% aqueous formic acid (10:30:60, v/v/v) at a flow rate of 0.3 ml/min. MS was detected by electrospray ion source negative ion mode and multiple reaction monitoring mode. The method was fully validated. The calibration curves for the five phenolic acids were linear in the given concentration ranges. The extraction recoveries, matrix effects, intra-day and inter-day precisions, and accuracies of the five analytes were all within acceptable limits. No significant difference of elimination half-life time (T1/2 ) of five analytes at three doses was observed. Area under the curve and peak concentration (Cmax ) of the five analytes demonstrated a linear increase in the doses with the linear correlation r of each analyte at three doses being greater than 0.915. It indicated that the pharmacokinetic behavior of is positively related to the dose in the range of 10-62.5 mg/kg.

Development of Hyphenated Techniques and Network Identification Approaches for Biotransformational Evaluation of Promising Antitubercular N-pyrrolyl hydrazide-hydrazone in Isolated Rat Hepatocytes.

Novel, rapid and precise RP-HPLC-DAD method was developed, validated and successfully applied for determination of metabolic changes of ethyl 5-(4-bromophenyl)-1-(3-(2-(2-hydroxybenzylidene)hydrazinyl)-3-oxopropyl)-2-methyl-1H-pyrrole-3-carboxylate (12b) in isolated rat hepatocytes. The analytes were detected by a simple DAD detector at 279 nm wavelength. A single-step extraction method was implemented to enable fast purification and extraction from cellular culture, resulting in a complete recovery. Thereafter, the method was adequately transferred to a LC-MS system for identification of unknown products. Additionally, network metabolism evaluation was performed to predict the structures of major metabolites with their isotope mass through BioTransformer 3.0. The data from the LC-MS analysis and the online server were compared for comprehensive identification. The results indicated formation of four metabolic products, obtained through processes of hydrolysis (12 and b), hydroxylation in the structure 12b (M1) and O-dealkylation (M2).

Mass Spectrometric Fingerprint Mapping Reveals Species-Specific Differences in Plant Polyphenols and Related Bioactivities.

Plant species show large variation in the composition and content of their tannins and other polyphenols. These large metabolites are not easy to measure accurately, but they are important factors for species bioactivity and chemotaxonomy. Here, we used an automated group-specific UHPLC-DAD-MS/MS tool to detect and quantify eight most common polyphenol groups in 31 chemically diverse plant species representing many types of growth forms and evolutionary ages. Ten replicate plants were used for each species and two polyphenol-related bioactivities, i.e., protein precipitation capacity and oxidative activity were measured in all samples as well. By the help of a novel 2D fingerprint mapping tool we were able to visualize the qualitative and quantitative differences between the species in hydrolysable tannins (galloyl and hexahydroxydiphenoyl derivatives), proanthocyanidins (procyanidins and prodelphinidins), flavonols (kaempferol, quercetin and myricetin derivatives) and quinic acid derivatives together with the two bioactivities. The highest oxidative activities were found with species containing ellagitannins (e.g., Quercus robur, Geranium sylvaticum, Lythrum salicaria and Chamaenerion angustifolium) or prodelphinidin-rich proanthocyanidins (e.g., Ribes alpinum, Salix phylicifolia and Lysimachia vulgaris). The best species with high protein precipitation capacity were rich in gallotannins (Acer platanoides and Paeonia lactiflora) or oligomeric ellagitannins (e.g., Comarum palustre, Lythrum salicaria and Chamaenerion angustifolium). These types of tools could prove their use in many types of screening experiments and might reveal even unusually active polyphenol types directly from the crude plant extracts.

Development of a Rapid LC-MS/MS Method for Simultaneous Quantification of Donepezil and Tadalafil in Rat Plasma: Its Application in a Pharmacokinetic Interaction Study after Oral Administration in Rats.

This study aimed to establish a simple and sensitive analytical method to simultaneously quantify donepezil (DPZ) and tadalafil (TAD) in rat plasma using lansoprazole (LPZ) as an internal standard (IS) by using liquid chromatography tandem mass spectrometry. The fragmentation pattern of DPZ, TAD, and IS was elucidated using multiple reaction monitoring in electrospray ionization positive ion mode for the quantification of precursor to production at m/z 380.1 → 91.2 for DPZ, m/z 390.2 → 268.1 for TAD, and m/z 370.3 → 252.0 for LPZ. The extracted DPZ and TAD from plasma using acetonitrile-induced protein precipitation was separated using Kinetex C18 (100 × 2.1 mm, 2.6 µm) column with a gradient mobile phase system consisting of 2 mM ammonium acetate and 0.1% formic acid in acetonitrile at a flow rate of 0.25 mL/min for 4 min. The selectivity, lower limit of quantification, linearity, precision, accuracy, stability, recovery, and matrix effect of this developed method was validated according to the guidelines of the U.S. Food and Drug Administration and the Ministry of Food and Drug Safety of Korea. The established method achieved acceptance criteria in all validation parameters, ensuring reliability, reproducibility, and accuracy, and was successfully implemented in a pharmacokinetic study on the co-administration of DPZ and TAD orally in rats.

Enzymatic degradation of RNA causes widespread protein aggregation in cell and tissue lysates.

Most proteins in cell and tissue lysates are soluble. We show here that in lysate from human neurons, more than 1,300 proteins are maintained in a soluble and functional state by association with endogenous RNA, as degradation of RNA invariably leads to protein aggregation. The majority of these proteins lack conventional RNA-binding domains. Using synthetic oligonucleotides, we identify the importance of nucleic acid structure, with single-stranded pyrimidine-rich bulges or loops surrounded by double-stranded regions being particularly efficient in the maintenance of protein solubility. These experiments also identify an apparent one-to-one protein-nucleic acid stoichiometry. Furthermore, we show that protein aggregates isolated from brain tissue from Amyotrophic Lateral Sclerosis patients can be rendered soluble after refolding by both RNA and synthetic oligonucleotides. Together, these findings open new avenues for understanding the mechanism behind protein aggregation and shed light on how certain proteins remain soluble.

Simple and high sample throughput LC/ESI-MS/MS method for bioequivalence study of prazosin, a drug with risk of orthostatic hypotension.

OBJECTIVE: The study aimed to develop a rapid, simple and sensitive LC/ESI-MS/MS method to measure prazosin concentration in human plasma and apply bedside sampling in bioequivalence study of two prazosin tablets to resolve the adverse effect of orthostatic hypotension. SIGNIFICANCE: The LC/ESI-MS/MS prazosin method was highly sensitive and selective. Bedside sampling reduced the orthostatic hypotension incidence and subject dropout rate. METHODS: After sample preparation, prazosin and terazosin (IS) were detected on mass spectrometer operating in multiple reaction monitoring mode using positive ionization. Mobile phase flow rate was set at 0.40 mL/min with sample run time of 1.75 min. The bioanalytical method was validated as per EMEA and FDA guidelines. Bedside sampling was performed in bioequivalence study for the first 4 h after dosing. The three primary pharmacokinetic parameters, Cmax, AUC0-t and AUC0-∞ and 90% confidence interval were determined. RESULTS: The small injection volume of 1 µL minimized instrumentation contamination and prolonged the analytical column lifespan. Linearity was obtained between 0.5 and 30.0 ng/mL, with coefficient of determination, r2 ≥ 0.99. The mean extraction recovery of prazosin and IS was >92%, with precision value (CV, %) ≤ 10.3%. Only two orthostatic hypotension adverse events were reported. The two prazosin formulations were found to be bioequivalent. CONCLUSION: The LC/ESI-MS/MS method has shown robustness and reliability exemplified by the incurred sample re-analysis result. Bedside sampling should be proposed for bioequivalence or pharmacokinetic studies of drugs demonstrating adverse event of orthostatic hypotension.

Fast and Sensitive HPLC-ESI-MS/MS Method for Etoricoxib Quantification in Human Plasma and Application to Bioequivalence Study.

Etoricoxib is a non-steroidal anti-inflammatory drug (NSAID) used to treat pain and inflammation. The objective of the current study was to develop a sensitive, fast and high-throughput HPLC-ESI-MS/MS method to measure etoricoxib levels in human plasma using a one-step methanol protein precipitation technique. A tandem mass spectrometer equipped with an electrospray ionization (ESI) source operated in a positive mode and multiple reaction monitoring (MRM) were used for data collection. The quantitative MRM transition ions were m/z 359.15 > 279.10 and m/z 363.10 > 282.10 for etoricoxib and IS. The linear range was from 10.00 to 4000.39 ng/mL and the validation parameters were within the acceptance limits of the European Medicine Agency (EMA) and Food and Drug Analysis (FDA) guidelines. The present method was sensitive (10.00 ng/mL with S/N > 40), simple, selective (K prime > 2), and fast (short run time of 2 min), with negligible matrix effect and consistent recovery, suitable for high throughput analysis. The method was used to quantitate etoricoxib plasma concentrations in a bioequivalence study of two 120 mg etoricoxib formulations. Incurred sample reanalysis results further supported that the method was robust and reproducible.

A Sensitive LC-MS/MS Method for the Simultaneous Determination of Two Thia-Analogous Indirubin N-Glycosides and Indirubin-3'-Monoxime in Plasma and Cell Culture Medium.

Indirubin was identified as an active component of Danggui Longhui Wan, an herbal mixture used in traditional Chinese medicine, and showed anticancer activity in clinical trials in patients with chronic leukemia. Investigations on the mechanisms of antitumor action of indirubins have mainly focused on the indirubin derivative indirubin-3'-monoxime (I3M). Meanwhile, antiproliferative and cytotoxic properties on cancer cells have also been demonstrated for several synthetic indirubin N-glycosides. In the present study, we demonstrate cytotoxic activity of the thia-analogous indirubin N-glycosides KD87 (3-[3'-oxo-benzo[b]thiophen-2'-(Z)-ylidene]-1-(ß-d-glucopyranosyl)-oxindole) and KD85 (3-[3'-oxo-benzo[b]thiophen-2'-(Z)-ylidene]-1-(ß-d-mannopyranosyl)-oxindole) against melanoma and squamous cell carcinoma cells as well as lung cancer and glioblastoma cells. The advanced state of preclinical studies on the effects of indirubins conducted to date underscores the need for pharmacokinetic data from cellular, animal, and human studies for which reliable quantification is required. Therefore, a sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated for the simultaneous measurement of KD87, KD85, and I3M in plasma and cell culture medium. Experimental conditions for sample preparation were optimized for human plasma protein precipitation and liquid-liquid extraction from plasma and cell culture medium. The methods were successfully validated in accordance with the U.S. Food and Drug Administration Bioanalytical Method Validation and evaluated for selectivity, sensitivity, matrix effect, recovery, carryover, calibration curve linearity, accuracy, precision, and stability. The applicability of the methods was demonstrated by the determination of KD87 in mouse plasma after prior intraperitoneal administration to mice.

Method development and validation of sildenafil, N-desmethylsildenafil and N1,N4-desmethylsildenafil by LC-MS/MS and its application.

This study aimed to develop an LC-MS/MS method for determining sildenafil and its metabolites N-desmethylsildenafil and N1,N4-desmethylsildenafil in human plasma and applying it to a pharmacokinetic study of sildenafil in healthy volunteers. Sildenafil-d8 was used as the internal standard. Plasma samples were pretreated via protein precipitation with acetonitrile. The extractives were then separated on an ACQUITY UPLC BEH C18 (50-mm × 2.1-mm, 1.7-µm) column using gradient elution. The aqueous and organic mobile phases were ammonium formate 2 mM supplemented with 0.1% formic acid in water and acetonitrile, respectively, and the flow rate was 0.3 mL/min. An electrospray ionization source was applied, and multiple reaction monitoring was operated in the positive mode with selective channels at m/z 475.30 â†’ 100.10, 461.20 â†’ 283.30, 483.30 â†’ 108.10, and 449.00 â†’ 283.00 for sildenafil, sildenafil-d8, N-desmethylsildenafil, and N1,N4-desmethylsildenafil, respectively. The linear calibration curves of sildenafil and its metabolites spanned 1.0-1000 ng/mL. The lower limit of quantification was 1.0 ng/mL. The extractive recovery of analytes from the biological matrix was more than 90% and the matrix effect complied with relevant provisions. The intra- and inter-day precisions of sildenafil and its metabolite were <10%. The intra- and inter-day accuracy of sildenafil, N-desmethylsildenafil, and N1,N4-desmethylsildenafil was more than 99%. The method is highly sensitive and selective, and it was successfully applied to the bioequivalence studies of 100-mg sildenafil citrate tablets in 40 healthy Chinese volunteers.

Simultaneous LC-MS/MS bioanalysis of alkaloids, terpenoids, and flavonoids in rat plasma through salting-out-assisted liquid-liquid extraction after oral administration of extract from Tetradium ruticarpum and Glycyrrhiza uralensis: a sample preparation strategy to broaden analyte coverage of herbal medicines.

Herbal medicines have historically been practiced in combinatorial way, which achieves therapeutic efficacy by integrative effects of multi-components. Thus, the accurate and precise measurement of multi bioactive components in matrices is inalienable to understanding the metabolism and disposition of herbal medicines. In this study, aiming to provide a strategy that improves analyte coverage, evaluation of six protocols employing sample pretreatment methods- protein precipitation (PPT), liquid-liquid extraction (LLE), sugaring-out-assisted liquid-liquid extraction (SULLE), and salting-out-assisted liquid-liquid extraction (SALLE)- was performed by LC-MS/MS using rat plasma and a mixture of alkaloid (evodiamine, rutaecarpine, dehydroevodiamine), terpenoid (limonin, rutaevin, obacunone), and flavonoid (liquiritin, isoliquiritin, liquiritigenin) standards isolated from Tetradium ruticarpum and Glycyrrhiza uralensis. These protocols were as follows: (1) PPT with methanol, (2) PPT with acetonitrile, (3) LLE with methyl tertiary-butyl ether-dichloromethane, (4) LLE with ethyl acetate-n-butanol, (5) SALLE with ammonium acetate, (6) SULLE with glucose. The results suggested that SALLE produced broader analyte coverage with satisfactory reproducibility, acceptable recovery, and low matrix interference. Then, sample preparation procedure of SALLE, chromatographic conditions, and mass spectrometric parameters were optimized, followed by method validation, showing that good sensitivity (LLOQ ≤ 1 ng mL-1), linearity (r ≥ 0.9933), precision (RSD ≤ 14.45%), accuracy (89.54~110.87%), and stability could be achieved. Next, the developed method was applied successfully to determine the pharmacokinetic behavior of the nine compounds in rat plasma after intragastric administration with an extract from Tetradium ruticarpum and Glycyrrhiza uralensis (Wuzhuyu-Gancao pair). Based on an extensive review and experiments, a sample preparation procedure that matches with LC-MS/MS technique and can get wider analyte coverage was outlined. The developed SALLE method is rapid, reliable, and suitable for bioanalysis of analytes with diverse polarity, which was expected to be a promising strategy for the pharmacokinetic studies of herbal medicines. Graphical abstract.

A highly sensitive, simple, and fast gas chromatography-mass spectrometry method for the quantification of serum short-chain fatty acids and their potential features in central obesity.

Short-chain fatty acids (SCFAs) are the main gut microbe metabolites, which have no more than six carbons. SCFAs are an emerging biomarker in metabolic diseases, including central obesity. Commonly, SCFAs are measured in fecal samples, where they are highly abundant, but here they do not reflect direct interactions with related organs. Serum SCFAs are assumed to be more associated with metabolic disease than fecal SCFAs, albeit at very low concentrations. The aim of the present study is to develop a highly sensitive, simple, and fast method for measuring six SCFAs in the serum by gas chromatography-mass spectrometry (GCMS). The serum is mixed with meta-phosphoric acid and 2,2-dimethylbutyric acid, followed by homogenization and centrifugation. Supernatant is then injected into the fused silica capillary column. The method is linear from 0.12-500 µmol/L for all SCFAs with an accuracy of 90-117%. The total coefficient of variation for precision ranges from 3.8 to 14.1%. A preliminary study is performed with 32 centrally obese subjects and 17 lean subjects. The mean values of all SCFAs, including acetic, propionic, isobutyric, butyric, isovaleric, and valeric acid, in the centrally obese subjects are significantly higher compared with lean subjects. A significant correlation also exists between all SCFAs, with the waist circumference indicating that serum SCFAs have potential features with respect to metabolic diseases, especially central obesity. The validated GCMS method provides highly sensitive, fast, simple, and reliable SCFA quantitation in the serum and demonstrates the potential features of circulating SCFAs in central obesity.

Simultaneous quantification of cyclosporin, tacrolimus, sirolimus and everolimus in whole blood by UHPLC-MS/MS for therapeutic drug monitoring.

The aim of this study was to develop and validate a UHPLC-MS/MS assay to quantify cyclosporin (CYC), tacrolimus (TAC), sirolimus (SIR) and everolimus (EVE) in human whole blood for therapeutic drug monitoring. Analytes were extracted from 50 µL human whole blood by protein precipitation. The separation of the drugs was performed on an Acquity UPLC BEH C18 column. Analytes were eluted with a mobile phase consisting of 2 mM ammonium acetate with 0.1% formic acid (v/v) in deionised water and 2 mM ammonium acetate with 0.1% formic acid (v/v) in methanol at a flow rate of 300 µL/min in gradient elution. The method performance was evaluated by analysing patient blood samples and/or external quality control samples [proficiency testing (PT) scheme]. The method was linear from 23.75 to 1094.0, 1.3 to 42.4, 1.3 to 47.0 and 1.2-41.6 µg/mL for CYC, TAC, SIR and EVE, respectively. The within- and between-assay reproducibility results were ˂ 11%. Results from PT and patient sample quantification were comparable to those obtained previously by an in-house validated method using protein precipitation and liquid-liquid extraction. This method showed good analytical performance for quantifying CYC, TAC, SIR and EVE in whole blood over their respective calibration ranges.

Tannin Content in Vitis Species Red Wines Quantified Using Three Analytical Methods.

Tannin content in red wines is positively correlated with astringency perception and wine grade; however, tannin quantification is one of the main challenges. In this study, tannin content was quantified using three analytical methods in commercial red wines from Vitis vinifera and interspecific cold-hardy hybrids including Marquette, Frontenac, and Petite pearl cultivars. Protein (PP) and methylcellulose precipitation (MCP) methods were compared to a HPLC-DAD method, which is based on the interaction between tannins and a hydrophobic surface (RPC). Frontenac wines were the poorest in tannins and Cabernet sauvignon wines were the richest regardless of the method used. In cold-hardy red wines, the tannin content was higher in Marquette with high alcohol content, which suggested that the tannins were extracted from seeds rather than skins. The high limit of quantification of the PP method and the presence of anthocyanin di-glucosides in cold-hardy wines were parameters suggesting that protein and methylcellulose precipitation methods were neither suitable nor reliable for the quantification of tannins in cold-hardy red wines. The tannin content quantified by RPC was positively correlated to tannin quantified by MCP, suggesting that the RPC method would be relevant for the quantification of tannins in red wines.

A Rapid and Simple UHPLC-MS/MS Method for Quantification of Plasma Globotriaosylsphingosine (lyso-Gb3).

Fabry disease (FD) is a rare X-linked lysosomal storage disorder caused by α-galactosidase A gene (GLA) mutations, resulting in loss of activity of the lysosomal hydrolase, α-galactosidase A (α-Gal A). As a result, the main glycosphingolipid substrates, globotriaosylceramide (Gb3) and globotriaosylsphingosine (lyso-Gb3), accumulate in plasma, urine, and tissues. Here, we propose a simple, fast, and sensitive method for plasma quantification of lyso-Gb3, the most promising secondary screening target for FD. Assisted protein precipitation with methanol using Phree cartridges was performed as sample pre-treatment and plasma concentrations were measured using UHPLC-MS/MS operating in MRM positive electrospray ionization. Method validation provided excellent results for the whole calibration range (0.25-100 ng/mL). Intra-assay and inter-assay accuracy and precision (CV%) were calculated as <10%. The method was successfully applied to 55 plasma samples obtained from 34 patients with FD, 5 individuals carrying non-relevant polymorphisms of the GLA gene, and 16 healthy controls. Plasma lyso-Gb3 concentrations were larger in both male and female FD groups compared to healthy subjects (p < 0.001). Normal levels of plasma lyso-Gb3 were observed for patients carrying non-relevant mutations of the GLA gene compared to the control group (p = 0.141). Dropping the lower limit of quantification (LLOQ) to 0.25 ng/mL allowed us to set the optimal plasma lyso-Gb3 cut-off value between FD patients and healthy controls at 0.6 ng/mL, with a sensitivity of 97.1%, specificity of 100%, and accuracy of 0.998 expressed by the area under the ROC curve (C.I. 0.992 to 1.000, p-value < 0.001). Based on the results obtained, this method can be a reliable tool for early phenotypic assignment, assessing diagnoses in patients with borderline GalA activity, and confirming non-relevant mutations of the GLA gene.