Liquid Chromatography-mass Spectroscopy/mass Spectroscopy Research Articles

Altered expression of MZB1 in periodontitis: A possible link to disease pathogenesis.

Our previous study explored the molecular signatures of generalized aggressive periodontitis (GAgP) using gingival tissues through omics-based-whole-genome transcriptomic analysis. This continuation study aimed to investigate the whole protein profiling of these gingival samples through liquid chromatography-mass spectroscopy/mass spectroscopy (LC-MS/MS) analysis and to validate the identified proteins through immunohistochemistry to provide further evidence for the quality of the results. In previous study, gene expression patterns were identified in gingival tissues from 23 GAgP and 25 control individuals. In the current study, comparative proteomic analysis was performed on isolated proteins from the same study groups using LC-MS/MS analysis. The data from the transcriptomics study published before and the proteomics data were integrated to reveal any common genes and proteins. Additionally, immunohistochemical analysis was conducted to further investigate the findings. The most upregulated proteins in patients compared to controls were ITGAM, AZU1, MMP9, BPI, UGGG1, MZB1, TRFL, PDIA6, PRDX4, and PLG. The top six pathways associated with these proteins were involved in innate immune system, post-translational protein phosphorylation, interleukin-4 and -13 signaling, toll-like receptors cascades, and extracellular matrix organization. Based on the integration and validation analysis of transcriptomics and proteomics data, as well as immunohistochemical analysis, MZB1 was identified as a shared gene and protein that were upregulated in the patients. MZB1 is a protein that is involved in the development of B cells and the production of antibodies. Its upregulation in periodontitis suggests that there may be a dysregulation of the immune response in this condition, and MZB1 may be a potent biomarker for periodontitis.

A Review on Hyphenated Techniques in Analytical Chemistry

Background: In chemical and pharmaceutical analysis, hyphenated techniques range from the combinations involving separation-separation, separation-identification and identification-identification techniques and are widely used nowadays, as they hold many advantages like fast accurate analysis, a higher degree of automation, higher sample throughput, better reproducibility, specificity and sensitivity. They also reduce contamination due to closed systems and offer simultaneous separation and quantification, leading to better analysis. Objective: Though many reviews have appeared on hyphenated analytical techniques till date, in the past decade, their use has increased manifold and therefore, we thought it imperative to review the latest progress in this field. In the present article, an attempt has been made to cover the latest information on various hyphenated techniques like LC-MS (Liquid Chromatography-Mass Spectroscopy), GC-MS (Gas Chromatography-Mass Spectroscopy), LC-IR (Liquid Chromatography-Infra-Red Spectroscopy), as well as, LC-MS-MS (Liquid Chromatography-Mass Spectroscopy-Mass Spectroscopy), LC-NMR-MS (Liquid Chromatography-Nuclear Magnetic Resonance-Mass Spectroscopy), etc. Conclusion: This review describes a total of seventeen different hyphenated techniques, comprising mainly of the combinations of chromatographic techniques with spectroscopic techniques. We have tried to cover the latest information on various double hyphenated techniques like LC-MS , LC-NMR, LC-IR, HPTLC-MS, HPTLC-IR, GC-MS, GC-IR, GC-TLC, GC-AES, MS-MS, CE-MS, GC-NMR, as well as, triple hyphenated techniques like LC-MS-MS, LC-NMR-MS, LC-UV-MS, GC-MS-MS, GC-IR-MS. Mainly the principle, instrumentation, applications, and advantages of each of the techniques are discussed in this review. Also, disadvantages of a few techniques have been mentioned.

A concise review on analytical profile of risperidone

Risperidone (RIS) is an atypical antipsychotic medication. This works by selectively antagonising serotonin 5HT2 and dopamine D2 receptors. It's used to treat schizophrenia and other mental illnesses. As per literature, RIS was first approved USA by Food and drug administration (FDA) in 1993. Therefore, the main objective of this analysis of RIS in pharmaceutical and biological formulation is in both qualitative and quantitative terms. In this review article, we have summarized UV/Vis spectroscopy, high-performance liquid chromatography (HPLC), High-performance thin-layer chromatography (HPTLC), Liquid chromatography-mass spectroscopy-mass spectroscopy (LC-MS/MS) etc. based methods for estimation of risperidone. In addition to that, we have discussed the bioanalytical methods for RIS analysis. In conclusion, this review article will help to research scholars for further method development for drug estimation in pharmaceutical dosage forms and biological fluids.

Characterization and Anticancer Activity from Gaharu (Aquilaria malaccensis) Stem Bark Extract

Aquilaria malaccensis is a plant species from the Thymeleaceae family. A. malaccensis contains secondary metabolites of phenolic groups, namely benzophenone, chromone, coumarin, xanthone, and flavonoids with various biological activities such as antioxidants and anticancer. This study aims to determine the potential of A. malaccensis stem bark extract as an anticancer and to determine the characteristics of secondary metabolite compounds contained in A. malaccensis stem bark extract. Extraction of the stem bark of A. malaccensis by maceration method in stages using n -hexane, ethyl acetate and methanol as solvents. The anticancer activity test used the microculture tetrazolium (MTT) assay against Murine Leukemia P-388 cells. Compound separation using thin layer chromatograph (TLC) and gravity column chromatography (CC) methods. Determination of chemical compounds using liquid chromatography mass spectroscopy/mass spectroscopy (LCMS/MS). The extracts of n -hexane, ethyl acetate, and methanol each had anticancer activity which was classified as active with an IC 50 value of 20.64, 26.61, and 14.49 µg/mL. Based on the results of separation and identification of compounds in methanol extract with TLC, CC, and LCMS/MS, methanol extract contains acetaminophen, aminosamptothesin, isophorone, diethyl phthalate, 3-hydroxy-6H-benzochromen-6-one, isolongifolenic, 2-(2-phenethyl) chromone, 4′,7-dihydroxy-2′,5-dimethoxyisoflavone, 4-hydroxy-3-(1-phenylpropyl)-2H-chromone-2-one; 6-methylchromone-2-carboxylic acid; 3,4-dimethyl-2- phenylmorpholine; and β-calacoren compounds.

Coupling biological and photocatalytic treatment of atrazine and tebuthiuron in aqueous solution

Pollutants, such as pesticides, find their way into wastewater treatment plants (WWTPs). WWTPs have been reported to be unable to fully remove these pesticides from water. Hence, research has been conducted in the development of methods to degrade them in wastewater. This work reports the coupling of biological and photocatalytic processes to particularly treat two model pesticides (atrazine and tebuthiuron) in aqueous solution. Liquid chromatography-mass spectroscopy/mass spectroscopy (LC-MS/MS) was used as an advanced analytical technique for the identification of both atrazine and tebuthiuron, and their degradation by products. The LCMS/MS data showed that atrazine and tebuthiuron were only removed substantially during their first treatment days. Based on LCMS/MS results, biodegradation of atrazine in the biological treatment section was via dealkylation to yield deethylatrazine (m/z 198), (2) dechlorination to yield N-isopropylammelide (m/z 156), and (3) deamination to yield cyanuric acid (m/z 129). There were no identified metabolites of tebuthiuron which suggested its structural stability, but a decrease was observed at the beginning of the treatment of the pollutant probably due to adsorption on the sludge. Photocatalysis further reduced the concentration of atrazine, while there was no significant decrease in tebuthiuron. The atrazine degradation pathways that were identified during photocatalysis studies were (1) degradation of atrazine (CIET, m/z 216) through the formation of hydroxyatrazine (OEIT, m/z 197), desethylhydroxyatrazine (OIAT, m/z 155), ammeline (OAAT, m/z 127) and cyanuric acid (OOOT, m/z 129), and (2) the degradation of atrazine, after the formation hydroxyatrazine (OEIT, m/z 197), could also proceed to form deethylatrazine (CIAA, m/z 198), followed by the formation of N-isopropylammelide (IAT, 156) and finally cyanuric acid (OOOT, m/z 129). The bio-photodegradation treatment process was efficient for the removal of atrazine but not for the removal of tebuthiuron.

Development and Validation of Liquid Chromatography- Mass Spectroscopy/Mass Spectroscopy Method for Quantitative Analysis of Naproxen in Human Plasma after Liquid-Liquid Extraction

Purpose: To determine naproxen levels in human plasma using a new liquid chromatography-Mass spectroscopy/Mass spectroscopy (LC-MS/MS) method that involves a simple and single step extraction procedure using low-cost reagents. Method: A novel liquid chromatography‐tandem mass spectrometry method for the quantitative determination of naproxen in human K2-EDTA plasma in negative ion mode was employed and validated using zidovudine as internal standard (IS). Sample preparation was accomplished by liquidliquid extraction technique. The eluted samples were chromatographed on Zorbax Eclipse XDB phenyl 4.6 ◊ 75 mm, 3.5 µm column (Agilent Technologies) using a mobile phase consisting of acetonitrile: 20 mM ammonium acetate (90:10 v/v).The injection volume was 15 µL and the total run time was 3.0 min. The method was validated for all parameters for naproxen. Results: The method showed selectivity and linearity over a concentration range of 500.1 ng/mL to 100028.5 ng/mL The validation data indicate precision and accuracy of 90 - 110 % and < 15 %), respectively, as well as recovery (80.63 %), stability (mostly stable) and carryover (0 %). Conclusion: A rapid and selective LC-MS/MS method for the quantification of naproxen in human plasma has been developed and can be used in therapeutic drug monitoring of this drug as well as in bioequivalence studies of the drug.

Protein Characterization of Extracted Water from Three Kinds of Edible Bird Nest Using SDS-PAGE CBB Staining and SDSPAGE Glycoprotein Staining and LC-MS/MS Analyses

The objective of this research is to see the comparison of protein characterization from three kinds of Indonesia edible bird nest as they have not been researched before. This research would give us overall picture on protein characteristics of the three Indonesian edible bird's nests, so it could show their activeness compared to the white edible bird nest. This research was a laboratory research with three treatments and five parameters. Treatments in this research was conducted on three kinds of edible bird nest, the white edible bird nest (Collocaliafuciphaga), the black edible bird nest (Collocalia maxima) and the swallow edible bird nest (Collocalialinchi). Parameters measured in this research were protein content, glycoprotein content, SDS- PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel) CBB (Coomassie brilliant blue) staining, SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel) glycoprotein staining and LC-MS/MS(Liquid Chromatography- mass Spectroscopy/mass spectroscopy) analysis. It was an explorative research. The method used was trial method with randomized complete design (protein and glycoprotein content) and descriptive method. The analyses results on protein and glycoprotein content from the three Indonesian edible bird nests were different and presumably due to their different kinds of feed. Protein characterization using SDS-PAGE CBB staining showed protein band high intensity on various molecular weights. They are 140.8; 64.8; 21.2 kDa for the white edible bird nest, 27.1; 21.2; 15.2 kDa for the black edible bird nest, and 140.8; 116; 36.2 kDa for the swallow edible bird nest. Glycoprotein characterization using SDS-PAGE glycoprotein staining showed different results; 140.8 and 64.8 kDa for the white edible bird nest; 140.8 and 116.0 kDa for the swallow edible bird nest; 28.4 and 105.3 kDa for the black edible bird nest. Analysis results using LC-MS/MS showed the presence of glycoprotein in the white edible bird nest but none was found in the black and the swallow edible bird nest.

Nested Case Control Study of Proteomic Biomarkers for Interstitial Lung Disease in Japanese Patients With Non–Small-Cell Lung Cancer Treated With Erlotinib: A Multicenter Phase IV Study (JO21661)

BackgroundInterstitial lung disease (ILD) is a serious adverse drug reaction associated with epidermal growth factor receptor tyrosine-kinase inhibitors (EGFR TKIs). Its risk factors are yet to be fully elucidated. We sought to identify proteomic biomarkers associated with ILD development in erlotinib-treated Japanese patients with non–small-cell lung cancer (NSCLC) to build predictive models. Patients and MethodsWe conducted a nested case-control study. The participants were patients with NSCLC enrolled in a phase IV study of erlotinib in whom ILD developed within 120 days after erlotinib administration. The controls were randomly selected patients without ILD from the overall study cohort who were also treated with erlotinib. Serum samples were obtained before the first administration of erlotinib and were assayed by liquid chromatography–mass spectroscopy/mass spectroscopy (LC-MS/MS). Logistic regression analysis was performed to identify the peptide and proteins associated with ILD. ResultsA total of 645 patients were enrolled in the cohort; 15 case patients and 64 controls were analyzed. When multiplicity was taken into account, we were unable to statistically verify any genuine association between individual markers and ILD. Investigation of the predictive power based on leave-one-out cross-validation (LOOCV) showed that the area under the receiver operating characteristic curve was 0.73 at a maximum. Additional analysis suggested that 3 proteins (C3, C4A/C4B, and APOA1) have a stronger association with ILD than do the other proteins tested. ConclusionWe were unable to demonstrate predictive serum protein markers for ILD development. However, C3, C4A/C4B, and APOA1 are worthy of further investigation.

Determination of Fatty Acid Ethyl Esters (FAEE) and Ethyl Glucuronide (EtG) in Hair: A Promising Way for Retrospective Detection of Alcohol Abuse During Pregnancy?

The retrospective detection of alcohol consumption during pregnancy is an important part of the diagnosis of the fetal alcohol syndrome. A promising way to solve this problem can be the determination of fatty acid ethyl esters (FAEE) or/and ethyl glucuronide (EtG) in hair of the mothers. In this article, the present state in analytical determination and interpretation of FAEE and EtG concentrations in hair are reviewed. Both FAEE and EtG are minor metabolites of ethanol and as direct alcohol markers very specific for alcohol. They are durably deposited in hair, which enables taking advantage of the long diagnostic time window of this sample material. In the last years, specific and sensitive methods for determination of both alcohol markers in hair were developed. Headspace solid phase microextraction in combination with gas chromatography-mass spectroscopy after hair extraction with an n-heptane/dimethylsulfoxide mixture proved to be a favorable technique for determination of four characteristic FAEE (ethyl myristate, ethyl palmitate, ethyl oleate, and ethyl stearate). EtG is extracted from hair by water and analyzed either by gas chromatography-mass spectroscopy with negative chemical ionization after cleanup with solid phase extraction and derivatization with pentafluoropropionic anhydride or by liquid chromatography-mass spectroscopy-mass spectroscopy. The detection limits of the single FAEE as well as of EtG are in the range of 1 to 10 pg/mg. FAEE as well as EtG were determined in a larger number of hair samples of teetotalers, social drinkers, patients in alcohol withdrawal treatment, and death cases with previous known heavy drinking. From the results, the following criteria were derived: strict abstinence is excluded or improbable at C FAEE >0.2 ng/mg or C EtG >7 pg/mg. Moderate social drinkers should have C FAEE <0.5 ng/mg and C EtG <25 pg/mg; above these values, alcohol abuse is probable. Until now, there has been no evaluation in context of FAS diagnosis; however, a successful application for this purpose can be expected from the good experience in driving ability examination.

Isolation and sequence of an interferon-tau-inducible, pregnancy- and bovine interferon-stimulated gene product 15 (ISG15)-specific, bovine ubiquitin-activating E1-like (UBE1L) enzyme.

Bovine (bov) interferon-stimulated gene product 15 (ISG15) is produced in the endometrium in response to conceptus-secreted interferon (IFN)-tau. ISG15 conjugates to endometrial proteins through an enzymatic pathway that is similar to ubiquitinylation. Ubiquitin-activating enzyme 1-like protein (UBE1L) initiates enzymatic conjugation by forming a thioester bond with ISG15, thus preparing it for transfer to the next series of enzymes. The bovUBE1L has not been described. We hypothesized that bovUBE1L was induced by pregnancy and IFN-tau in the endometrium. A 110-kDa protein was purified from bovine endometrial (BEND) cells based on affinity with recombinant (r) glutathione S-transferase (GST)-ISG15. This protein was digested in gel with trypsin. Seven peptides were purified using HPLC, sequenced using liquid chromatography-mass spectroscopy-mass spectroscopy and found to share 43-100% identity with human UBE1L. The full-length bovUBE1L cDNA was isolated from a BEND cell cDNA library, sequenced, and found to share 83% identity with human UBE1L cDNA. Northern blot revealed two mRNAs that were detected in greater (P<0.05) concentrations in endometrium from Day 17-21 pregnant versus nonpregnant cows. Western blots using antihuman UBE1L antibody revealed a similar pattern of pregnancy-associated expression of UBE1L protein in the uterus. The bovUBE1L mRNA was localized, using in situ hybridization, primarily to glandular and luminal epithelium, with more diffuse localization to stroma of the endometrium from pregnant cows. Because bovUBE1L was purified through its interaction with rGST-ISG15 and shares significant amino acid and cDNA sequence identity with human UBE1L, it is concluded that it mediates conjugation of ISG15 to uterine proteins in response to the developing and attaching conceptus.

Influence of Formaldehyde Impurity in Polysorbate 80 and PEG‐300 on the Stability of a Parenteral Formulation of BMS‐204352: Identification and Control of the Degradation Product

The purpose of this study was to identify a degradation product formed in the clinical parenteral formulation of BMS‐204352, investigate the role of excipients in its formation, and develop a strategy to minimize/control its formation. The degradant was identified as the hydroxy methyl derivative (formaldehyde adduct, BMS‐215842) of the drug substance based upon liquid chromatography/mass spectroscopy (LC/MS), liquid chromatography/mass spectroscopy/mass spectroscopy (LC/MS/MS), nuclear magnetic resonance (NMR), and chromatographic comparison to an authentic sample of hydroxymethyl degradation product, BMS‐215842. An assay method for the detection of formaldehyde based on HPLC quantitation of formaldehyde dinitrophenylhydrazone was developed to quantitate its levels in various Polysorbate 80 and PEG 300 excipient lots. A direct relationship between the levels of formaldehyde in the excipients and the formation of the hydroxymethyl degradant was found. To confirm the hypothesis that the formaldehyde impurity in these two excipients contributed to the formation of the hydroxymethyl degradant, several clinical formulation lots were spiked with formaldehyde equivalent to 1, 10, and 100 mg/g of BMS‐204352. A correlation was found between the formaldehyde level and the quantity of the hydroxymethyl degradant formed upon storage at 5 and 25°C. From these experiments, a limit test on the formaldehyde content in polysorbate 80 and PEG 300 can be set as part of a strategy to limit the formation of the degradation product.

Induction of Plasma Vitellogenin Synthesis by the Commercial Fish Diets in Male Goldfish(Carassius auratus) and Dietary Phytoestrogens.

In this study, we analyzed contents of phytoestrogens (genistein, daidzein, equol, and coumestrol) in two commercial fish diets [a diet for trout (TD) and a diet for ornamental carp (CD)] using Liquid Chromatography-Mass Spectroscopy/Mass Spectroscopy (LC-MS/MS), and these contents were compared with that of a casein-based formulated fish diet (FD) which does not contain soya bean or fish meal. We also analyzed phytoestrogen contents in commercial infant casein- and soya bean-based diets. The contents of phytoestorogens were generally high in CD, TD, and soya milk, and low or non-detectable in FD and casein-based milks. Among these samples, CD showed the highest phytoestrogen contents: genistein, 390800 ng/g; daidzein, 416800 ng/g; coumestrol, 1325 ng/g; equol, 6.4 ng/g. We also determined the estrogenic activity of the fish diets using male goldfish by measuring plasma vitellogenin (VTG) levels as a biomarker of estrogen exposure. When male goldfish were fed one of these diets for 31 days, plasma VTG was detected in CD-fed fish (78.01 ± 48.18 μg/ml) and TD-fed fish (3.51 ± 3.83 μg/ml), whereas plasma VTG was not detected in FD-fed fish (less than 0.040 μg/ml). These results indicate that the commercial fish diets examined contain a large amount of phytoestrogens and showed estrogenic activity that were strong enough to induce VTG production in male goldfish. It is necessary to eliminate estrogenic substances other than test chemicals in the screening test system for estrogenic endocrine-disrupting chemicals (EDCs). Since the formulated diet developed in the present study contain less phytoestrogens than the commercial fish diets and has low estrogenic activity, it is suggested that VTG production using male goldfish in combination with the low estrogen fish diet is a good in vivo system for evaluation of estrogenic effects of EDCs.

Validity of Dual-Label Stable Isotopic Protocols and Urinary Excretion Ratios to Determine Folate Bioavailability from Food

We tested the hypothesis that some cereal-product vehicles may reduce fortificant bioavailability below 85% and examined the feasibility of using single dose, dual-label, non-saturation protocols for studying bioavailability based on urinary excretion ratios (UER; % oral 13C6 isotope dose excreted in intact folate/% i.v. 2H4 isotope dose excreted). Fifteen females received 225 micrograms oral folate (capsules, fortified bran flakes, and fortified white bread), mainly as 13C6-PteGlu, followed by i.v. injection of 100 micrograms 2H4-PteGlu. UERs were used as the primary index of absorption. Urinary folate was cleaved to p-aminobenzoylglutamic acid, dervivatized and determined by gas chromatography/mass spectroscopy (GC/MS). The UER mean (95%CI) for folic acid was 2.18 (1.2-3.8) at 48 hours and as these were greater than 1.0, it was concluded that oral and i.v. isotopes of folic acid are handled differently by the body. Compared to the 48 hour UER for folic acid, UERs for white bread and bran flakes were 0.71 and 0.37, respectively, thus indicating some matrix inhibition of absorption. Consideration should be given to the choice of cereal-based fortification vehicles in order to maximize bioavailability. Plasma enrichment of folate can be measured using LC/MSMS (liquid chromatography/mass spectroscopy-mass spectroscopy) but seems unfeasible with the GC/MS method.

Evaluation of intraocular pharmacokinetics and toxicity of prinomastat (AG3340) in the rabbit.

To determine the ocular pharmacokinetics, physiological and histological effects of prinomastat (a matrix metalloprotease inhibitor), a total of seventy-seven eyes of New Zealand White rabbits received intravitreous and subtenon injections of prinomastat or of acidified water vehicle as control, Doses of 0.5 mg in 0.05 mL of prinomastat or acidified water were used for intravitreal injection. For the subtenon injections, doses of 5 mg prinomastat in 0.5 mL of acidified water were administered in the superotemporal quadrant. Intraocular pharmacokinetics were determined by analyzing vitreous samples at different postinjection time points using Liquid Chromatography-Mass Spectroscopy/Mass Spectroscopy (LC-MS/MS). The toxicity was evaluated by biomicroscopy, electroretinography (ERG), pneumatonometry, and histology. No toxicity was found with either administration method. At day 14 after intravitreal injection, levels of prinomastat in the vitreous and choroid were 1.4 ng/mg and 7.8 ng/mg, respectively. The retinal levels of prinomastat were 22 ng/mg at 24 hr and dropped below 1 ng/mg at 48 hr. Prinomastat remained well above minimum effective concentration in the choroid for at least four weeks after a single intravitreal injection, suggesting that local intravitreal injection may have potential in treating choroidal neovascularization.