Liquid Chromatography Coupled With Mass Spectrometry Analysis Research Articles

A new methodology combining QCM-D and proteomic profiling enables characterization of protein adsorption on 2D surfaces.

One of the critical features of biomedical material design is controlling the plasma protein adsorption to modulate the material behavior in biological media. Protein adsorption is highly influenced by the material surfaces and the proteins present in the biological medium. Thus, it is necessary to study protein-surface interactions that eventually take place on nanomaterials introduced into the body by the use of human plasma. However, very little information is available about human plasma interaction with planar surfaces under physiological conditions. Due to the limitation of the current characterization techniques to investigate the complicated interaction between the complex milieu of plasma proteins and planar materials, most efforts have focused on single proteins. To face this challenge, we have developed a new methodology based on the combination of quartz crystal microbalance with dissipation monitoring (QCM-D) and liquid chromatography coupled with mass spectrometry (LC-MS) to obtain information about protein-surface interactions on planar surfaces. First, QCM-D allowed us to determine the adsorbed protein mass and layer thickness. After detaching the proteins by a surfactant treatment, LC-MS analysis revealed the proteomic profile. Here, we have investigated three base materials, polystyrene (PS), gold (Au), and silica (SiO2) with or without precoating and compared the protein profiles.

An Optimised Monophasic Faecal Extraction Method for LC-MS Analysis and Its Application in Gastrointestinal Disease.

Liquid chromatography coupled with mass spectrometry (LC-MS) metabolomic approaches are widely used to investigate underlying pathogenesis of gastrointestinal disease and mechanism of action of treatments. However, there is an unmet requirement to assess faecal metabolite extraction methods for large-scale metabolomics studies. Current methods often rely on biphasic extractions using harmful halogenated solvents, making automation and large-scale studies challenging. The present study reports an optimised monophasic faecal extraction protocol that is suitable for untargeted and targeted LC-MS analyses. The impact of several experimental parameters, including sample weight, extraction solvent, cellular disruption method, and sample-to-solvent ratio, were investigated. It is suggested that a 50 mg freeze-dried faecal sample should be used in a methanol extraction (1:20) using bead beating as the means of cell disruption. This is revealed by a significant increase in number of metabolites detected, improved signal intensity, and wide metabolic coverage given by each of the above extraction parameters. Finally, we addressed the applicability of the method on faecal samples from patients with Crohn's disease (CD) and coeliac disease (CoD), two distinct chronic gastrointestinal diseases involving metabolic perturbations. Untargeted and targeted metabolomic analysis demonstrated the ability of the developed method to detect and stratify metabolites extracted from patient groups and healthy controls (HC), highlighting characteristic changes in the faecal metabolome according to disease. The method developed is, therefore, suitable for the analysis of patients with gastrointestinal disease and can be used to detect and distinguish differences in the metabolomes of CD, CoD, and HC.

Evaluation of the Determination of Dabigatran, Rivaroxaban, and Apixaban in Lupus Anticoagulant-Positive Patients.

Background: The effect of direct oral anticoagulants (DOAC) on laboratory tests dependent on the production of their targets, factor IIa and factor Xa, is a well-known problem and can cause both false positive and negative results. In particular, the situation in patients who develop lupus anticoagulant (LA) antibodies is highly complex. To evaluate the effectiveness of DOAC therapy in lupus-positive patients, 31 samples were enrolled in this retrospective study. All patient samples were spiked with three types of DOAC (dabigatran, DABI; rivaroxaban, RIVA; and apixaban, API) in a concentration that significantly influenced the screening test for LA and thus can mask the presence of LA. Subsequently, the DOAC was always unbound by the DOAC-Stop procedure. DOAC levels before and after binding were determined by functional assays, followed by liquid chromatography coupled with mass spectrometry (LC-MS) analysis. Methods: The determination of DOAC levels was performed by direct thrombin assay and determination of anti-Xa activity with specific calibration as functional tests for DABI and xabans (API and RIVA). To determine concentration levels of API, DABI, and RIVA, our in-house LC-MS method was used. Results: The results of LA-positive samples show significant differences between functional tests and the LC-MS method both before and after DOAC binding. Conclusions: The acute findings of the presence of LA-type antibodies fundamentally affects the determination of DOAC by functional tests, and in this case, it is necessary to use LC-MS analysis to determine the true value. If patients treated with DOAC develop LA of medium and higher titers, we do not recommend checking DOAC levels with functional tests.

Simultaneous Estimation of Aflatoxins (B1, B2, G1 and G2) by Liquid Chromatography Coupled with Mass Spectrometry (LC-MS) in Corn Samples

Aflatoxins produced by Aspergillus flavins and Aspergillus pasasiticus have been investigated in the corn (maize) collected in different locations in Andhra Pradesh state, India. In the present study, 24 corn samples collected from different harvest, storage and dumping areas were analyzed for detection of aflatoxin contamination by liquid chromatography coupled with mass spectrometry (LC-MS). Mixed aflatoxin working standard solutions with a concentration of 10, 20, 30, 40, 50 and 60 ng/mL were prepared and followed by instrumental analysis and the retention times of the sample chromatograms were compared with the standard aflatoxins B1, B2, G1 and G2 retention times in order to determine the contamination. The findings revealed that among 24 collected samples, only 10 samples have been found positive for aflatoxins contamination when subjected to LC-MS analysis. Co-occurrence of aflatoxin was identified in the most of the samples. Even though 10 samples were tested positive total aflatoxin content in the most positive are found within the acceptable limits (30 μg/kg) and samples collected at dumping areas (S4, S8 and S10) show the presence of exceeding quantity than the limits described by FSSAI. Total aflatoxin content in the most positive were found within the acceptable limits (30 μg/kg) and the samples collected at dumping areas S4 (40.9 μg/kg), S8 (53.2 μg/kg) and S10 (71.86 μg/kg) had shown exceeding limits. The sample S7 collected from the storage was also found positive of presence of aflatoxin G2- 13.49 μg/kg and B2-21.14 μg/kg with total aflatoxin of 34.65 μg/kg. High contamination of these dumping areas may lead to contamination of storage points nearer to these areas. Present results showed that the corn samples at dumping areas were found to be contaminated with the Aspergillus flavins and Aspergillus pasasiticus.

Metabolomics in posttraumatic stress disorder: Untargeted metabolomic analysis of plasma samples from Croatian war veterans

Posttraumatic stress disorder (PTSD) is a severe, multifactorial and debilitating neuropsychiatric disorder, which can develop in a subset of individuals as a result of the exposure to severe stress or trauma. Such traumatic experiences have a major impact on molecular, biochemical and cellular systems, causing psychological and somatic alterations that affect the whole organism. Although the etiology of PTSD is still unclear, it seems to involve complex interaction between various biological genetic and environmental factors. Metabolomics, as one of the rapidly developing “omics” techniques, might be a useful tool for determining altered metabolic pathways and stress-related metabolites as new potential biomarkers of PTSD. The aim of our study was to identify metabolites whose altered levels allow us to differentiate between patients with PTSD and healthy control individuals. The study included two cohorts. The first, exploratory, group included 50 Croatian veterans with PTSD and 50 healthy control subjects, whereas a validation group consisted of 52 veterans with PTSD and 52 control subjects. The metabolomic analysis of plasma samples was conducted using liquid chromatography coupled with mass spectrometry (LC-MS), as well as gas chromatography coupled with mass spectrometry (GC-MS). The LC-MS analysis determined significantly different levels of two glycerophospholipids, PE(18:1/0:0) and PC(18:1/0:0), between control subjects and PTSD patients in both cohorts. The altered metabolites might play a role in multiple cellular processes, including inflammation, mitochondrial dysfunction, membrane breakdown, oxidative stress and neurotoxicity, which could be associated with PTSD pathogenesis.

Evaluation of the Antioxidant Activity of Nigella sativa L. and Allium ursinum Extracts in a Cellular Model of Doxorubicin-Induced Cardiotoxicity.

Natural products black cumin—Nigella sativa (N. sativa) and wild garlic—Allium ursinum (AU) are known for their potential role in reducing cardiovascular risk factors, including antracycline chemotherapy. Therefore, this study investigates the effect of N. sativa and AU water and methanolic extracts in a cellular model of doxorubicin (doxo)-induced cardiotoxicity. The extracts were characterized using Ultraviolet-visible (UV-VIS) spectroscopy, Fourier-transform infrared (FT-IR) spectroscopy, Liquid Chromatography coupled with Mass Spectrometry (LC-MS) and Gas Chromatography coupled with Mass Spectrometry (GC-MS) techniques. Antioxidant activity was evaluated on H9c2 cells. Cytosolic and mitochondrial reactive oxygen species (ROS) release was evaluated using 2′,7′-dichlorofluorescin-diacetate (DHCF-DA) and mitochondria-targeted superoxide indicator (MitoSOX red), respectively. Mitochondrial membrane depolarization was evaluated by flow cytometry. LC-MS analysis identified 12 and 10 phenolic compounds in NSS and AU extracts, respectively, with flavonols as predominant compounds. FT-IR analysis identified the presence of carbohydrates, amino acids and lipids in both plants. GC-MS identified the sulfur compounds in the AU water extract. N. sativa seeds (NSS) methanolic extract had the highest antioxidant activity reducing both intracellular and mitochondrial ROS release. All extracts (excepting AU methanolic extract) preserved H9c2 cells viability. None of the investigated plants affected the mitochondrial membrane depolarization. N. sativa and AU are important sources of bioactive compounds with increased antioxidant activities, requiring different extraction solvents to obtain the pharmacological effects.

Derivatization of Methylglyoxal for LC-ESI-MS Analysis-Stability and Relative Sensitivity of Different Derivatives.

The great research interest in the quantification of reactive carbonyl compounds (RCCs), such as methylglyoxal (MGO) in biological and environmental samples, is reflected by the fact that several publications have described specific strategies to perform this task. Thus, many reagents have also been reported for the derivatization of RCCs to effectively detect and quantify the resulting compounds using sensitive techniques such as liquid chromatography coupled with mass spectrometry (LC-MS). However, the choice of the derivatization protocol is not always clear, and a comparative evaluation is not feasible because detection limits from separate reports and determined with different instruments are hardly comparable. Consequently, for a systematic comparison, we tested 21 agents in one experimental setup for derivatization of RCCs prior to LC-MS analysis. This consisted of seven commonly employed reagents and 14 similar reagents, three of which were designed and synthesized by us. All reagents were probed for analytical responsiveness of the derivatives and stability of the reaction mixtures. The results showed that derivatives of 4-methoxyphenylenediamine and 3-methoxyphenylhydrazine—reported here for the first time for derivatization of RCCs—provided a particularly high responsiveness with ESI-MS detection. We applied the protocol to investigate MGO contamination of laboratory water and show successful quantification in a lipoxidation experiment. In summary, our results provide valuable information for scientists in establishing accurate analysis of RCCs.

Chemical characterization and bioactivity of Trichosanthes dioica edible shoot extract

Present investigation was aimed to evaluate the traditional use of edible part of soft shoots of Trichosanthes dioica Roxb. (Cucurbitaceae) as an antidiabetic in mice model. In addition, antioxidant activity and chemical profiling of this plant part were also performed to support its observed activity. The extract was subjected to oral glucose tolerance test in normal and alloxan induced diabetic mice to explore its antihyperglycemic activity. Antioxidant capacity was analyzed by a number of in vitro assays. Quantification of bioactive polyphenols was done by HPLC. Liquid Chromatography coupled with Mass Spectrometry (LCMS) was used to identify chemical constituents present in the extract. Total polyphenol and flavonoids content were found in significant quantity. In DPPH radical scavenging assay the IC50 value of the extract was found to be 148.62 µg/mL. Reducing power of the extract was comparable with that of butylatedhydroxytoluene (BHT). HPLC analysis indicated that quercetin, rutin, p-coumaric acid and kaempferol were the major bioactive polyphenols present in the extract. Further chemical profiling using LCMS analysis was identified a total of nine compounds with different chemical classes. In OGTT, extract (400 mg/kg BW) showed a 31.13% decrease (p < 0.05) in blood glucose levels at 30 min compared to the normal control. In alloxan induced diabetic mice the extract at the doses of 200 mg and 400 mg/kg, showed significant decrease (p < 0.05) of blood glucose level compared to diabetic control. The extract showed oral glucose tolerance potential and antioxidant capacity which might be due to the presence of different compounds such as quercetin, rutin, kaempferol, oleanolic acid, β-sitosterol. The results support the scientific basis of it ethnobotanical uses in traditional medicinal practices of Bangladesh.

Derivatization-based High-throughput Bioanalysis by LC-MS.

Liquid chromatography coupled with mass spectrometry (LC-MS) is one of the most prominent analytical techniques due to its inherent selectivity and sensitivity. LC-MS is currently the first choice for high-throughput bioanalysis due to the advancements in MS instruments and the analytical software. Based on this situation, we are developing various types of derivatization reagents, including chiral reagents for MS and/or MS/MS detection. These developed reagents are adopted for the detection of biomarker candidates related to diseases. The biomarker candidates include not only achiral molecules, but also chiral ones. Although determining the already-identified chiral molecules is relative easy, it is very difficult to identify and/or determine unknown enantiomer(s) in real samples. To solve this difficulty, we proposed a new strategy to identify unknown enantiomeric biomarkers related to diseases. This review paper deals with the development of derivatization reagents for amines and carboxylic acids in LC-MS analysis and their application to bioanalysis.

A microbiological method for determining serum levels of broad spectrum β-lactam antibiotics in critically ill patients

BackgroundRecent studies show that suboptimal blood levels of β-lactam antibiotics are present in intensive care unit (ICU) patients. A common reference method for assessing drug concentrations is liquid chromatography coupled with mass-spectrometry (LC-MS) which is highly accurate but rarely available outside reference centres. Thus, our aim was to develop a microbiological method for monitoring β-lactam antibiotic serum levels which could be used at any hospital with a microbiological laboratory. MethodsThe method was developed as a 96-well broth microdilution format to assess the concentrations of cefotaxime (CTX), meropenem (MER), and piperacillin (PIP). Patient serum containing antibiotics were diluted in suspensions of bacteria with known minimal inhibitory concentrations (MICs). Serum antibiotic concentrations were calculated by dividing the MIC with the dilution factor at which the serum inhibited growth of the bacterial suspension. Serum (n=88) from ICU patients at four hospitals in south-east Sweden were analysed and compared to LC-MS analysis. ResultsThe overall accuracy and precision for spiked samples and patient samples was within the pre-set target of ±20.0% for all drugs. There was a significant correlation between the microbiological assay and LC-MS for the patient samples (CTX: r=0.86, n=31; MER: r=0.96, n=11; PIP: r=0.88, n=39) and the agreement around the clinical cut-off for CTX (4.0mg/l), MER (2.0mg/l) and PIP (16.0mg/l) was 90%, 100% and 87%, respectively. ConclusionThe microbiological method has a performance for determination of serum levels of meropenem, piperacillin and cefotaxime suitable for clinical use. It is an inexpensive method applicable in any microbiology laboratory.

Antinociceptive effects of the aqueous and methanol extracts of the leaves of Pittosporum mannii Hook. F. (Pittosporaceae) in mice

Ethnopharmacological relevancePittosporum mannii (Pittosporaceae) is used in Africa traditional medicine to treat various ailments including pain and inflammation. Aim of the studyThe present work was undertaken to evaluate the antinociceptive effects of the aqueous (AEPM) and methanol (MEPM) extracts from the leaves of Pittosporum mannii. MethodsHigh performance liquid chromatography coupled with mass spectrometry (LCMS) was used for the phytochemical analysis of AEPM prepared as decoction and MEPM prepared as cold maceration. The in vitro cytotoxicity of AEPM and MEPM were evaluated on Artemia salina larvae. AEPM and MEPM antinociceptive effects were evaluated at the doses of 35, 75, 150 and 300mg/kg given orally, against pain induced by acetic acid, formalin, hot plate, capsaicin and glutamate. The rota rod test was also performed at the same doses. To determine the mechanism of action of these extracts, their antinociceptive effects were tested in animals pretreated with yohimbine (α2-adrenergic antagonist), atropine (muscarinic antagonist) or naloxone (an opioids antagonist). ResultThe LCMS analysis showed that both extracts contain pittovidoside and 1-O-rhamnopyranosyl-23-acetoxyimberbic acid 29-methyl ester, the aqueous extract being more concentrated. Oral administration of both extracts significantly reduced pain symptoms induced by acetic acid, formalin, capsaicin, glutamate and hot plate. The antinociceptive effect of AEPM was significantly inhibited by yohimbine, atropine and naloxone while these inhibitors tend to potentiate the activity of MEPM. Both extracts have no effect on Rota rod test. AEPM and MEPM showed respective LC50 of 2.44 and 0.70mg/ml on Artemia larvae and were therefore, considered non-toxic. ConclusionThese results indicate that AEPM and MEPM possesses analgesic effects with different mechanism of action. Although effects of both extracts may involve TRPV1 receptors and glutamatergic pathway, AEPM may in addition, interact with alpha-adrenergic, muscarinic and opioidergic pathways that are not involve in the effects of MEPM.

Antinociceptive properties of the aqueous and methanol extracts of the stem bark of Petersianthus macrocarpus (P. Beauv.) Liben (Lecythidaceae) in mice

Ethnopharmacological relevanceAqueous maceration from the stem barks of Petersianthus macrocarpus (P. Beauv.) Liben (Lecythidaceae) is taken orally in the central Africa for the management of various ailments, including pain. Aim of the studyThis work was carried out to evaluate in mice, the antinociceptive effects of the aqueous and methanol extracts of the stem bark of P. macrocarpus. Materials and methodsThe chemical composition of the aqueous and methanol extracts prepared as cold macerations was determined by high performance liquid chromatography coupled with mass spectrometry (LCMS). The antinociceptive effects of these extracts administered orally at the doses of 100, 200 and 400mg/kg were evaluated using behavioral pain model induced by acetic acid, formalin, hot-plate, capsaicin and glutamate. The rotarod test was also performed at the same doses. The oral acute toxicity of both extracts was studied at the doses of 800, 1600, 3200 and 6400mg/kg in mice. ResultThe LCMS analysis revealed the presence of ellagic acid as the major constituent in the methanol extract. Both extracts of P. macrocarpus significantly and dose dependently reduced the time and number of writhing induced by acetic acid. They also significantly inhibited the two phases of formalin-induced pain. These effects were significantly inhibited by a pretreatment with naloxone, except for the analgesic activity of the methanol extract at the earlier phase. In addition, nociception induced by hot plate, intraplantar injection of capsaicin or glutamate was significantly inhibited by both extracts. Acute toxicity test showed no sign of toxicity. ConclusionThese results demonstrate that aqueous and methanol extracts of P. macrocarpus are none toxic substances with good central and peripheral antinociceptive effects that are at least partially due to the presence of ellagic acid. These extracts may induce their antinociceptive effect by interfering with opioid, capsaicin and excitatory amino acid pathways.

Determination of Kanamycin Plasma Levels Using LC-MS and Its Pharmacokinetics in Patients

Purpose: The objectives of the study were: (1) to determine kanamycin plasma concentrations using liquid chromatography coupled with mass spectrometry (LC-MS), (2) to investigate kanamycin pharmacokinetics (PK) in patients with multi-drug resistant tuberculosis (MDR-TB), (3) to find out whether HIV infection, kidney dysfunction and antiretroviral drugs influence kanamycin PK. Methods: The study was designed as a non-randomized study involving male and female HIV- positive and HIV negative patients admitted for MDR-TB treatment. Blood samples were collected before (baseline) and ½, 1, 2, 4, 8 and 24 hours after intramuscular injection of kanamycin. LC-MS was used to quantify kanamycin plasma concentrations. Results: Thirty one patients including 13 HIV (+) participated in the study. The lower limit of detection and lower limit of quantification of kanamycin were 0.06 μg/ml and 0.15 μg/ml respectively. Kanamycin PK parameters were described and there was no significant difference between HIV-positive and HIV-negative patients. A statistical significant difference (p=0.0126) was found in the renal function in HIV - positive and HIV - negative patients. However, this difference did not affect kanamycin elimination. No interactions have been identified between antiretroviral drugs and kanamycin. Conclusion: LC-MS analysis method is highly specific and highly sensitive in the detection and quantification of kanamycin plasma concentrations. Kanamycin PK in patients with MDR-TB was described. Due to a limited number of patients, we cannot rule out any influence of HIV - infection, renal impairment and antiretroviral drugs on kanamycin pharmacokinetics. The relationship between the area under the curve of kanamycin free plasma concentrations (fAUC) and its minimum inhibitory concentrations (MIC) on M.tuberculosis isolated from the sputum of each patient should be assessed. Therefore, kanamycin free plasma concentrations and MIC should be determined

Complete Mapping of a Cystine Knot and Nested Disulfides of Recombinant Human Arylsulfatase A by Multi-Enzyme Digestion and LC-MS Analysis Using CID and ETD

Cystine knots or nested disulfides are structurally difficult to characterize, despite current technological advances in peptide mapping with high-resolution liquid chromatography coupled with mass spectrometry (LC-MS). In the case of recombinant human arylsulfatase A (rhASA), there is one cystine knot at the C-terminal, a pair of nested disulfides at the middle, and two out of three unpaired cysteines in the N-terminal region. The statuses of these cysteines are critical structure attributes for rhASA function and stability that requires precise examination. We used a unique approach to determine the status and linkage of each cysteine in rhASA, which was comprised of multi-enzyme digestion strategies (from Lys-C, trypsin, Asp-N, pepsin, and PNGase F) and multi-fragmentation methods in mass spectrometry using electron transfer dissociation (ETD), collision induced dissociation (CID), and CID with MS(3) (after ETD). In addition to generating desired lengths of enzymatic peptides for effective fragmentation, the digestion pH was optimized to minimize the disulfide scrambling. The disulfide linkages, including the cystine knot and a pair of nested cysteines, unpaired cysteines, and the post-translational modification of a cysteine to formylglycine, were all determined. In the assignment, the disulfide linkages were Cys138-Cys154, Cys143-Cys150, Cys282-Cys396, Cys470-Cys482, Cys471-Cys484, and Cys475-Cys481. For the unpaired cysteines, Cys20 and Cys276 were free cysteines, and Cys51 was largely converted to formylglycine (>70%). A successful methodology has been developed, which can be routinely used to determine these difficult-to-resolve disulfide linkages, ensuring drug function and stability.

Identification of Maillard reaction induced chemical modifications on Ara h 1

The Maillard reaction is a non-enzymatic glycation reaction between proteins and reducing sugars that alters peanut allergens during thermal processing. We are characterizing Maillard reaction dependent modifications on recombinant Ara h 1 protein. Recombinant Ara h 1 produced in E. coli was purified by ion exchange chromatography and incubated in the presence of reducing sugars at elevated temperature in a simulated roasting model. Samples containing modified Ara h 1 were removed at various times and analyzed by immunoblot with Maillard reaction-specific antibodies and liquid chromatography coupled with mass spectrometry (LC-MS). LC-MS samples were reduced by the addition of DTT, alkylated with iodoacetamide, and digested with protease. The resulting peptide mixture was analyzed on an Agilent nano-LC fitted with Agilent 1200 nano-pumps and a 6500 Q-TOF LC. Spectral data from the analysis were evaluated with Spectrum Mill software. We find that recombinant Ara h 1 has increased reaction with anti-carboxymethyl lysine and other Maillard reaction specific antibodies. We observed greater than 80% coverage of the recombinant Ara h 1 protein by LC-MS. Several Maillard reaction-dependent modifications on the Ara h 1 protein were detected by the LC-MS analysis including carboxymethylation, carboxyethylation, malondialdehyde, and pyrrolysine. Some of the modifications were found to lie within previously characterized linear IgE epitopes of Ara h 1. We have identified Maillard reaction induced chemical modifications on Ara h 1, and these modifications may help explain the elevated recognition by IgE and the increased protein stability of peanut proteins after thermal processing.