Liquid Chromatography-electrospray Ionization-tandem Mass Research Articles

LC-MS/MS Method for Simultaneous Determination of Linarin and Its Metabolites in Rat Plasma and Liver Tissue Samples: Application to Pharmacokinetic and Liver Tissue Distribution Study After Oral Administration of Linarin.

Linarin, a flavone glycoside, is considered to be a promising natural product due to its diverse pharmacological activities. Recently, it has been brought into focus for its potential to treat liver failure. In this study, a rapid and sensitive liquid chromatography electrospray-ionization tandem mass spectrometry (LC–MS/MS) method was developed and validated for the simultaneous determination of linarin and its three metabolites (acacetin, apigenin, and p-hydroxy benzaldehyde) in plasma and liver tissue samples of normal rats and rats with d-galactosamine (d-GalN)-induced liver injury. After liquid–liquid extraction (LLE) with ethyl acetate, chromatographic separation of the four analytes was achieved using an ACQUITY UPLC BEH-C18 (1.7 μm, 2.1 × 50 mm) with a mobile phase of 0.01% formic acid in methanol and 0.01% formic acid at a flow rate of 0.3 mL/min. The detection was accomplished on a tandem mass spectrometer via an electrospray ionization (ESI) source by multiple reaction monitoring (MRM) in the negative ionization mode. The method had a good linearity over the concentration range of 1.00–200 ng/mL for linarin and its metabolites. The validated method was successfully applied to the pharmacokinetic and liver tissue distribution study of linarin and its metabolites after a single oral administration of linarin (90 mg/kg) to rats.

Elevated Plasma Ceramides Are Associated With Higher White Matter Hyperintensity Volume-Brief Report.

Sphingolipids, including S1P (sphingosine-1-phosphate) and ceramides, have been associated with vascular tone, blood pressure regulation, cardiovascular outcomes, and mortality. However, the relationship between plasma sphingolipids and cerebrovascular disease has not been examined. We aimed to assess the cross-sectional association between plasma sphingolipids and white matter hyperintensity (WMH) volume, which is a marker of cerebrovascular disease. Approach and Results: We included 588 participants (302 men and 286 women), aged 60 to 93, enrolled in the population-based Mayo Clinic Study of Aging who had MRI and plasma sphingolipids at the same study visit. Fasting plasma was obtained, and ceramides and S1P were assayed using liquid chromatography-electrospray ionization tandem mass spectrometry. Fluid-attenuated inversion recovery was used to measure WMH volume, defined as percent total intracranial volume. We used linear regression to cross-sectionally examine the relationships between plasma sphingolipids and WMH; both were log-transformed. In multivariable analyses adjusting for age, sex, and hypertension, higher levels of ceramide C16:0 (b [95% CI]=0.24 [0.02-0.45]) and the ceramide ratios C16:0_24:0 (b [95% CI]=0.30 [0.12-0.48]) and C24:1_24:0 (b [95% CI]=0.24 [0.07-0.41]) were associated with a higher WMH volume. A higher ceramide score was also associated with higher WMH volume (b [95% CI]=0.03 (0.01-0.04]). We did not observe any association between S1P and WMH volume. Higher plasma ceramide C16:0 and 2 specific ceramide ratios (C16:0_24:0 and C24:1_24:0) are associated with greater WMH volumes, independent of hypertension, suggesting their utility for measurement of cerebrovascular disease.

Nω -(Carboxymethyl)arginine Is One of the Dominant Advanced Glycation End Products in Glycated Collagens and Mouse Tissues.

Advanced glycation end products (AGEs) accumulate in proteins during aging in humans. In particular, the AGE structure Nω-(carboxymethyl)arginine (CMA) is produced by oxidation in glycated collagen, accounting for one of the major proteins detected in biological samples. In this study, we investigated the mechanism by which CMA is generated in collagen and detected CMA in collagen-rich tissues. When various protein samples were incubated with glucose, the CMA content, detected using a monoclonal antibody, increased in a time-dependent manner only in glycated collagen, whereas the formation of Nε-(carboxymethyl)lysine (CML), a major antigenic AGE, was detected in all glycated proteins. Dominant CMA formation in glycated collagen was also observed by electrospray ionization-liquid chromatography-tandem mass spectrometry (LC-MS/MS). During incubation of glucose with collagen, CMA formation was enhanced with increasing glucose concentration, whereas it was inhibited in the presence of dicarbonyl-trapping reagents and a metal chelator. CMA formation was also observed upon incubating collagen with glyoxal, and CMA was generated in a time-dependent manner when glyoxal was incubated with type I–IV collagens. To identify hotspots of CMA formation, tryptic digests of glycated collagen were applied to an affinity column conjugated with anti-CMA. Several CMA peptides that are important for recognition by integrins were detected by LC-MS/MS and amino acid sequence analyses. CMA formation on each sequence was confirmed by incubation of the synthesized peptides with glyoxal and ribose. LC-MS detected CMA in the mouse skin at a higher level than other AGEs. Furthermore, CMA accumulation was greater in the human aorta of older individuals. Overall, our study provides evidence that CMA is a representative AGE structure that serves as a useful index to reflect the oxidation and glycation of collagen.

Dose-dependent exposure profile and metabolic characterization of notoginsenoside R1 in rat plasma by ultra-fast liquid chromatography-electrospray ionization-tandem mass spectrometry.

Notoginsenoside R1 (NGR1 ), a diagnostic protopanaxatriol-type (ppt-type) saponin in Panax notoginseng, possesses potent biological activities including antithrombotic, anti-inflammatory, neuron protection and improvement of microcirculation, yet its pharmacokinetics and metabolic characterization as an individual compound remain unclear. The aim of this study was to investigate the exposure profile of NGR1 in rats after oral and intravenous administration and to explore the metabolic characterization of NGR1 . A simple and sensitive ultra-fast liquid chromatographic-tandem mass spectrometric method was developed and validated for the quantitative determination of NGR1 and its major metabolites, and for characterization of its metabolic profile in rat plasma. The blood samples were precipitated with methanol, quantified in a negative multiple reaction monitoring mode and analyzed within 6.0 min. Validation parameters (linearity, precision and accuracy, recovery and matrix effect, stability) were within acceptable ranges. After oral administration, NGR1 exhibited dose-independent exposure behaviors with t1/2 over 8.0 h and oral bioavailability of 0.25-0.29%. A total of seven metabolites were characterized, including two pairs of epimers, 20(R)-notoginsenoside R2 /20(S)-notoginsenoside R2 and 20(R)-ginsenoside Rh1 /20(S)-ginsenoside Rh1 , with the 20(R) form of saponins identified for the first time in rat plasma. Five deglycometabolites were quantitatively determined, among which 20(S)-notoginsenoside R2 , ginsenoside Rg1 , ginsenoside F1 and protopanaxatriol displayed relatively high exploration, which may partly explain the pharmacodynamic diversity of ginsenosides after oral dose.

Systemic availability of lipophilic organic UV filters through dermal sunscreen exposure

BackgroundChemical UV filters are common components in sunscreens and cosmetic products and used to protect the skin against harmful effects of sunlight like sunburn. However, the effectiveness of sunscreens in the prevention of skin cancer is in some parts still controversial. Meanwhile, questions about negative effects of the chemical UV filters on human health arise and request an effective risk assessment. Real-life exposure data in humans after application of these products are still rare. Thus, we explored whether and to what extent UV filters are absorbed through the skin into the human body. Material and methodsPlasma and urine samples from 20 healthy volunteers were collected before, during and after a real-life exposure scenario (1st application: 2 mg/cm2; 2nd and 3rd (after 2 and 4 h): 1 mg/cm2 each) using a commercial sunscreen formulation for one day. These samples were analyzed for their content of the currently prominent UV filters octocrylene and avobenzone as well as 2-cyano-3,3-diphenylacrylic acid (CDAA) as the main octocrylene metabolite by using different liquid chromatography electrospray-ionization tandem mass spectrometric procedures. ResultsFollowing dermal sunscreen exposure, avobenzone, octocrylene and CDAA reached concentrations up to 11 μg/L, 25 μg/L and 1352 μg/L in plasma. In urine detection rates of avobenzone and octocrylene were low while CDAA showed a high detection rate and reached up to 5207 μg/g creatinine. Kinetic models could be fitted for octocrylene and CDAA in plasma and CDAA in urine. Concentration peaks were reached between 10 and 16 h after first application and half-life periods were in the range of 1.5 to 2 days. The lipophilic UV filter octocrylene and its metabolite CDAA showed a much slower elimination than other more hydrophilic UV filters. Concordantly, the metabolite CDAA in particular showed a markedly increased renal excretion over the whole sampling period and indicated high internal exposure to OC. DiscussionReal-life sunscreen usage leads to considerable bioavailability of organic UV filters and their metabolites which is rarely seen for other environmental exposures. A combined monitoring of the parent compound and its metabolites is important to fully address internal exposure to the UV filter in humans. Considering the kinetic profiles a prolonged systemic release due to depot formation in skin and a potential accumulation through multi-day exposure is presumed. High in-vivo loads call for a critical toxicological assessment of the UV filters and their metabolites.

Total glucosides of paeony (TGP) extracted from Radix Paeoniae Alba exerts neuroprotective effects in MPTP-induced experimental parkinsonism by regulating the cAMP/PKA/CREB signaling pathway

Ethnopharmacological relevanceThe totally-amounted glucosides of paeony (TGP), which are made up of paeoniflorin, albiflorin, oxypaeoniflorin as well as benzoylpaeoniflorin, constitute the Baishao’ actively-working component extracted from Radix Paeonia alba employed in conventional oriental medicine aiming to treat cerebrovascular disorders, such as Parkinson's disease. However, its pharmacologic mechanism is not clear. Aim of the studyThe initial investigation was made on TGP's neuroprotective effects on PD of the mouse model based on 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) as well as the identification of potential involvement of a molecular signaling pathway. Materials and methodsThe evaluation of the behavioral damage as well as neurotoxicity in mice was made through MPTP. Spontaneous motor activity test, as well as a test of Rota-rod on mice was employed for the measurement of bradykinesia symptom. Additionally, liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS-MS) works as the determiner of the main monoamine neurotransmitters dopamine (DA) along with its metabolites 3, 4-dihydroxyphenylacetic acid (DOPAC) as well as homovanillic acid (HVA) based on mouse hippocampus connected with the anti-Parkinson's disease like effect of TGP. Besides, the measurement of the effects of TGP treatment on the expressions level of TH, DAT, a-synuclein, p-CREBS133 as well as apoptosis influence was made with the help of western-blot assay with apoptosis-related markers such as Bax and Bcl-2. ResultsThe results showed that TGP treatment lessened the behavior-based loss shown “in the spontaneous motor activity as well as the potential of falling to rotarod test”. In addition, we found that pretreatment with TGP markedly improved motor coordination, striatal dopamine and its metabolite levels. Furthermore, pretreatment of TGP conducted the protection for dopaminergic neurons with the prevented MPTP-induced reductions within the tyrosine hydroxylase (TH), substantia nigra dopaminergic transporter (DAT), as well as increasing α-synuclein protein levels with transformed dopamine catabolism as well as inhibited dopamine turnover. Besides, TGP treatment helped reversed apoptosis signaling molecules Bcl-2/Bax’ reduction; meanwhile improving p-CREBS133 the factor of growth signaling in the substantia nigra’ decrease. ConclusionThese results suggested that TGP can enhance dopaminergic neuron's cell survival in the SNpc in virtue of the activated cAMP/PKA/CREB factor of growth on inhibiting the pathway of second messenger apoptosis as well. In conclusion, the current findings indicate TGP is expected to be a new cure for PD.

Toxicokinetics of urinary 2-ethylhexyl salicylate and its metabolite 2-ethyl-hydroxyhexyl salicylate in humans after simulating real-life dermal sunscreen exposure.

Chemical UV filters are common components in sunscreens and cosmetic products. The question of adverse health risks is not completely resolved, partly owing to lacking human data from dermal exposure, which are essential for sound risk assessment. Therefore, we investigated the urinary toxicokinetics of 2-ethylhexyl salicylate (EHS) after a 1-day dermal real-life sunscreen application scenario. Twenty human volunteers were dermally exposed to a commercial sunscreen for 9h under real-life conditions (2mg/cm2 body surface area; double re-application; corresponding to 3.8g EHS). Urine samples were analyzed for EHS and one of its specific metabolites 2-ethyl-5-hydroxyhexyl salicylate (5OH-EHS) using a two-dimensional liquid chromatographic electrospray-ionization tandem mass spectrometric procedure. EHS and 5OH-EHS were excreted after sunscreen application and reached up to 525µg/g and 213µg/g creatinine, respectively. The toxicokinetic models showed concentration peaks between 7 and 8h after first application. First-phase terminal half-lives were 8-9h. For 5OH-EHS, a second-phase terminal half-life could be determined (87h). EHS and 5OH-EHS showed a faster elimination with 70-80% of the overall excretion occurring within 24h after application compared to more lipophilic UV filters. Cumulative excreted amounts over 24h reached up to 334µg EHS and 124µg of 5OH-EHS. Simulated real-life sunscreen use for 1day leads to the bioavailability of the UV filter EHS in humans. The kinetic profiles with a prolonged systemic availability indicate a skin depot and make accumulation during consecutive multi-day exposure likely.

Highly sensitive analytical method for herbicide clopyralid residue in cattle manure compost with ultraperformance liquid chromatography tandem mass spectrometry.

A micro liquid-liquid extraction has been applied to sample preparation in the current authorized method for clopyralid in compost. The method rendered matrix effects practically negligible during determination with ultraperformance liquid chromatography-electrospray ionization tandem mass spectrometry with an improved limit of quantification of 0.7 µg/kg dry weight. Moreover, it had good accuracy and reproducibility. Therefore, the method is proposed as a highly effective routine analytical technique for investigating the actual status of clopyralid residue in compost.

Ziziphora taurica subsp. taurica: Analytical Characterization and Biological Activities.

The Lamiaceae family comprises many flowering plants classified into about 236 genera. The genus Ziziphora is one of the well-known genera of this family and its species are important in different fields of pharmaceutical, chemical, traditional, and folk medicines. The phytochemicals present in Ziziphora include monoterpenic essential oils, triterpenes, and phenolic substances. The aim of this paper was to study the phytochemical profile of Ziziphora taurica subsp. taurica and compare and evaluate the biological activities of its ethyl acetate (ZTT-EtOAc), methanolic (ZTT-MeOH), and aqueous (ZTT-W) extracts based on their enzyme inhibition and antioxidant capacities. Determination of total phenolic (TPC) and total flavonoid (TFC) contents as well as biological activities were determined using spectrophotometric procedures. Subsequently, the individual phenolic compounds were detected by liquid chromatography–electrospray ionization–tandem mass spectrometry (LC–ESI–MS/MS). In total, twenty-two different phenolic compounds were identified, including apigenin, ferulic acid, and luteolin which were the most common. ZTT-MeOH extract showed the best antioxidant activity, whereas ZTT-EtOAc extract was the most effective against tyrosinase and α-amylase. Ziziphora taurica subsp. taurica represents a potential source of natural compounds with positive effects on human health.

Micro-extraction by packed sorbent combined with UHPLC-ESI-MS/MS for the determination of prostanoids and isoprostanoids in dried blood spots

This work presents a reliable analytical procedure combining micro-extraction by packed sorbent (MEPS) and ultra-high performance liquid chromatography-electrospray ionization tandem mass spectrometry to determine 8-iso prostaglandin F2α, 8-iso prostaglandin E2 and prostaglandin E2 in dried blood spots (DBSs). To reach this goal, we optimized a fast semi-automated MEPS procedure for the clean-up and pre-concentration of the analytes extracted from a single DBS (50 μL) by a 70:30 v/v methanol:water mixture. Limits of detection of about 20 pg mL−1, satisfactory recoveries (90–110%) and very good intra- and inter-day precisions (RSD ≤10%) were obtained for all the analytes. The innovative addition of internal standards on the filter paper before DBS sampling allowed to compensate changes in the amount of analyte during storage. Since prostanoids and isoprostanoids are biomarkers involved in the pathogenesis and progression of many diseases (e.g. ductal patency, diabetic nephropathy, and acute lung injury), our analytical method offers interesting diagnostic and prognostic opportunities in the medical field. The present method is currently used for the analysis of such biomarkers in DBSs from preterm newborns collected in the clinical setting.

Lysophosphatidylcholine is a Major Component of Platelet Microvesicles Promoting Platelet Activation and Reporting Atherosclerotic Plaque Instability.

Microvesicles (MVs) are small cell-derived vesicles, which are mainly released by activated cells. They are part of a communication network delivering biomolecules, for example, inflammatory molecules, via the blood circulation to remote cells in the body. Platelet-derived MVs are known to induce vascular inflammation. Research on the mediators and mechanisms of their inflammatory effects has attracted major interest. We hypothesize that specific lipids are the mediators of vascular inflammation caused by platelet-derived MVs. Liquid chromatography electrospray ionization-tandem mass spectrometry was used for lipid profiling of platelet-derived MVs. Lysophosphatidylcholine (LPC) was found to be a major component of platelet-derived MVs. Investigating the direct effects of LPC, we found that it induces platelet activation, spreading, migration and aggregation as well as formation of inflammatory platelet-monocyte aggregates. We show for the first time that platelets express the LPC receptor G2AR, which mediates LPC-induced platelet activation. In a mouse model of atherosclerotic plaque instability/rupture, circulating LPC was detected as a surrogate marker of plaque instability. These findings were confirmed by matrix-assisted laser desorption ionization imaging, which showed that the LPC concentration of human plaques was highest in vulnerable plaque regions. LPC is a major component of platelet-derived MVs and via its interaction with G2AR on platelets contributes to platelet activation, spreading, migration and aggregation and ultimately to vascular inflammation. Circulating LPC reports on atherosclerotic plaque instability in mice and is significantly increased in unstable areas of atherosclerotic plaques in both mice and humans, linking LPC to plaque instability.

Development and Validation of an HPLC-ESI/MS/MS Method for the Determination of Amoxicillin, Its Major Metabolites, and Ampicillin Residues in Chicken Tissues.

A method for the simultaneous analysis of amoxicillin (AMO), amoxicillin metabolites, and ampicillin residues in edible chicken muscle, liver, and kidney samples via high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI/MS/MS) was developed and verified. The extraction and purification procedures involved the extraction of the sample using a liquid-liquid extraction method with acetonitrile to eliminate the proteins. The chicken tissue extract was then injected directly onto an HPLC column coupled to a mass spectrometer with an ESI(+) source. The HPLC-ESI/MS/MS method was validated according to specificity, sensitivity, linearity, matrix effects, precision, accuracy, decision limit, detection capability, and stability, as defined by the European Union and Food and Drug Administration. The linearity was desirable, and the determination coefficients (r2 values) ranged from 0.9968 and 0.9999. The limits of detection and limits of quantification were 0.10–2.20 μg/kg and 0.30–8.50 μg/kg, respectively. The decision limits were 57.71–61.25 μg/kg, and the detection capabilities were 65.41–72.50 μg/kg, and the recoveries of the four target analytes exceeded 75% at the limits of quantification and exceeded 83% at 25, 50, and 100 μg/kg (n = 6 at each level), confirming the reliability of this method for determining these analytes and providing a new detection technology. For real sample analysis, this experiment tested 30 chicken tissue samples, only one chicken muscle, liver, and kidney sample were contaminated with 5.20, 17.45, and 7.33 μg/kg of AMO values, respectively, while other target compounds were not detected in the 30 tested chicken tissue samples.

Content of Phenolic Compounds and Antioxidant Activity of New Gluten-Free Pasta with the Addition of Chestnut Flour.

Chestnut fruit abounds in carbohydrates, proteins, unsaturated fatty acids, fiber, polyphenolic compounds, as well as vitamins and micronutrients, that are behind the health-promoting properties of this plant. The purpose of the discussed research was to obtain innovative gluten-free pasta from rice and field bean flour enriched with a various addition of chestnut flour. Regarding the studied pasta, the following were determined: the content of free phenolic acids, total polyphenols, and antioxidant properties. Chromatographic analysis (HPLC-ESI-MS/MS (high-performance liquid chromatography-electrospray ionization tandem mass spectrometry)) revealed a wide variety of phenolic acids. In a sample with 20% and higher content of chestnut flour, as many as 13 acids were detected. Isoferulic acid prevailed. The total content of free phenolic acids and total polyphenols increased along with the increasing chestnut content. Moreover, in most cases, the content of individual acids increased with the addition of chestnut flour. Besides, the antioxidant activity was positively correlated with the addition of chestnut fruit flour, the content of free phenolic acids, and total polyphenols. Our research has demonstrated that our innovative gluten-free pasta, with the addition of chestnut flour, has a potential to be a source of polyphenolic compounds, including free phenolic acids, that are valuable for human health.

Sphingolipidomics Investigation of the Temporal Dynamics after Ischemic Brain Injury.

Sphingolipids (SPLs) have been proposed as potential therapeutic targets for strokes, but no reports have ever profiled the changes of the entire range of SPLs after a stroke. This study applied sphingolipidomic methods to investigate the temporal and individual changes in the sphingolipidome including the effect of atorvastatin after ischemic brain injury. We conducted sphingolipidomic profiling of mouse brain tissue by liquid chromatography-electrospray ionization tandem mass spectrometry at 3 h and 24 h after 1 h of middle cerebral artery occlusion (MCAO), and SPL levels were compared with those of the Sham control group. At 3 h post-MCAO, ceramides (Cers) exhibited an increase in levels of long-chain Cers but a decrease in very-long-chain Cers. Moreover, sphingosine, the precursor of sphingosine-1-phosphate (S1P), decreased and S1P increased at 3 h after MCAO. In contrast to 3 h, both long-chain and very-long-chain Cers showed an increased trend at 24 h post-MCAO. Most important, the administration of atorvastatin improved the neurological function of the mice and significantly reversed the SPL changes resulting from the ischemic injury. Furthermore, we used plasma samples from nonstroke control and stroke patients at time points of 72 h after a stroke, and found a similar trend of Cers as in the MCAO model. This study successfully elucidated the overall effect of ischemic injury on SPL metabolism with and without atorvastatin treatment. The network of SPL components that change upon ischemic damage may provide novel therapeutic targets for ischemic stroke.

Reactive carbonyl compounds, carbonyl stress, and neuroinflammation in methyl alcohol intoxication

Methyl alcohol intoxications are characterized by high lethality and high prevalence of serious visual and brain damage in survivors. The mechanisms of toxic brain damage are complex and the role of carbonyl stress has not been studied yet. We measured the acute and follow-up concentrations of reactive carbonyl compounds in patients with acute methyl alcohol intoxication. Blood samples were collected from 28 subjects hospitalized with confirmed methyl alcohol intoxication and from 36 subjects who survived poisoning 2 years after discharge. Serum concentrations of C6–12 reactive aldehydes were measured by liquid chromatography–electrospray ionization–tandem mass spectrometry. The acute concentrations of all measured reactive aldehydes were higher than the follow-up concentrations: 36.4 ± 4.8 vs. 21.6 ± 5.2 ng cm−3 for C6; 38.9 ± 5 vs. 17.0 ± 2.0 ng cm−3 for C7; 18.8 ± 3.9 vs. 4 ± 0 cm−3 for C8; 36.5 ± 3.9 vs. 19.0 ± 3.0 ng cm−3 for C9; 6.1 ± 0.4 vs. 4.0 ± 0.5 ng cm−3 for C10; 13.6 ± 3.0 vs. 3.7 ± 0.6 ng cm−3 for C11; and 7.8 ± 0.4 vs. 4.7 ± 0.4 ng cm−3 for C12 (all p < 0.001). The patients who survived the intoxication had higher concentration of reactive carbonyl compounds than those who died: 38.6 ± 5.9 vs. 28.3 ± 1.7 ng cm−3 for C6 (p = 0.002); 20.7 ± 4.7 vs. 11.8 ± 1.2 ng cm−3 for C8 (p = 0.001); 37.7 ± 4.8 vs. 31.8 ± 3.8 ng cm−3 for C9 (p = 0.042); and 7.9 ± 0.6 vs. 7.3 ± 0.5 ng cm−3 for C12 (p = 0.022). A significant association was present between severity of metabolic acidosis, anion gap, and the acute concentration of measured biomarkers: r = − 0.39; p = 0.046 for C6; r = − 0.42; p = 0.035 for C7; r = − 0.48; p = 0.012 for C8; r = − 0.39; p = 0.046 for C9; and r = − 0.47; p = 0.015 for C11. The acute concentration of C6–C12 reactive aldehydes positively correlated with the acute serum concentration of leukotrienes (all p < 0.05). Acute elevation of serum concentration of reactive carbonyl compounds suggests that carbonyl stress is involved in the mechanisms of leukotriene-mediated neuroinflammatory response to methyl alcohol-induced toxic brain damage.

METHOD DEVELOPMENT AND VALIDATION FOR THE ESTIMATION OF AMPRENAVIR IN HUMAN PLASMA BY LIQUID CHROMATOGRAPHY–ELECTROSPRAY IONIZATION–TANDEM MASS SPECTROMETRY

Objective: The main aim of the present study was to develop a sensitive liquid chromatography–electrospray ionization–tandem mass spectrometric technique for the quantitation of amprenavir in human plasma.&#x0D; Methods: Chromatographic separation was achieved on a reversed-phase Symmetry C18 (50 mm×4.6 mm, 3.5 μm) column with isocratic elution by acetonitrile and 0.1% v/v formic acid in the ratio of 90:10 v/v as mobile phase. Chromatographic peaks were resolved with 0.7 ml/min flow rate. Drug was extracted with ethyl acetate solvent by liquid–liquid extraction method. Monitoring of transition of m/z 506.2 and 71.0 for amprenavir and 628 and 421 for methyl-indinavir was made on multiple reaction monitoring.&#x0D; Results: Calibration curve of amprenavir was linear over 1–600 ng/ml concentration range with regression coefficient (r2) value of &gt;0.99. The % relative standard deviation values were &lt;8.5% for interday and intraday precision and accuracy. The method has excellent recovery, and the percentage recovery values of lower quality control (QC), median QC, and higher QC samples were 101.86%, 102.8%, and 99.28%, respectively.&#x0D; Conclusion: The drug was stable for more time at variable stability conditions, and method was successfully applicable to regular analysis of amprenavir in biological matrices.

Simultaneous quantification of five lignans from Schisandra chinensis in various tissues of rats

In this research study, a rapid, sensitive, and specific high-performance liquid chromatography–electrospray ionization–tandem mass spectrometry (HPLC–ESI–MS/MS) method was established and validated, in regard to the simultaneous quantification of five sedative and hypnotic lignans (schisandrin, schisandrol B, schisantherin A, deoxyschisandrin, and schisandrin B) in various tissues of rats (including heart, liver, spleen, lung, and kidney). The purpose of the study was to clarify the tissue distribution of the total lignans extract of Schisandra chinensis (SC). Then, the analytes were separated on a MERCK Purospher STAR LP C18 column (250 mm × 4.6 mm, 5 μm), with a mobile phase consisting of 0.05% (v/v) formic acid acetonitrile, and 0.05% (v/v) formic acid water, and a flow rate of 1 mL/min. All of the calibration curves of the five components showed good linearity (r &gt; 0.9950), with ranges of 4.8 to 1920 ng/mL for analytes. The intra-day and inter-day precisions (relative standard deviation [RSD] %) were within 13.76% for all of the analytes. The average recoveries of the five analytes were greater than 85.23%, and the mean value of the matrix effect ranged from 82.3% to 93.4%. The five analytes were confirmed to be stable during the storage, preparation, and analytic procedures. The major target tissues of the total lignans extract of the SC in the rats were the livers and kidneys.

Liquid chromatography tandem mass spectrometry for the simultaneous determination of metformin and pioglitazone in rat plasma: Application to pharmacokinetic and drug-drug interaction studies

Failure to attain and sustain long term glycemic control is an ongoing challenge in diabetes therapy. The trend to use a combined therapy and the risk of drug-drug interaction (DDI) are elevated and thus the need for sensitive analytical methods is of great significance. Herein, a simple, robust, and sensitive reverse phase high performance liquid chromatography–electrospray ionization-tandem mass spectrometry (ESI-MS/MS) method for simultaneous determination of metformin (MET) and pioglitazone (PGT) in rat plasma using canagliflozin (CAN) as internal standards (IS) was developed and fully validated. Prior Chromatographic separation on an Agilent Eclipse Plus C18 (4.6 × 100 mm, 3.5 μm) using gradient mobile phase system consisting of ammonium formate pH 4.5 and acetonitrile at a flow rate of 0.5 mL min−1, within a run time of 14 min, the antidiabetic drugs were extracted from rat plasma using acetonitrile-induced protein precipitation technique. Multiple reaction monitoring in positive ion mode was used for quantitation of precursor to production at m/z 130.1 → 71.0 & 60 for MET, 357.2 → 134.2 for PGT, and 462.16 → 191.1 for CAN. Method linearity was obeyed in the range of 1 to 5000 and 1 to 2500 ng mL−1 for MET and PGT, respectively. The developed method was validated in terms of accuracy, precision, selectivity, recovery, matrix effects, and stability as per US-FDA bioanalytical guidelines and successfully applied to clinical pharmacokinetic and DDI studies with a single oral administration of target compounds. The peak plasma concentrations (Cmax) and area under the concentration-time curve (AUC) of MET was significantly influenced by the concomitant administration of PGT at equal concentration and vice versa. PGT affected the absorption and elimination rate of MET via inhibition of organic cationic transporter (OCT). Molecular modeling study revealed the significant interaction of PGT with OCT. A potential DDI in type 2 diabetic patient receiving chronic treatment with MET and PGT deserves further attention and study to improve drug therapy and prevent adverse effects.

In vitro residual activities in 20 variants of phenylalanine hydroxylase and genotype-phenotype correlation in phenylketonuria patients

Phenylketonuria (PKU), caused by phenylalanine hydroxylase (PAH) gene variants, is a common autosomal inherited metabolic disease. So far, 1111 PAH variants have been revealed. The residual activity of the PAH variants is the key determinant of the metabolic phenotype and BH4 responsiveness in PKU patients.In this study, the spectrum of PAH variants in 1083 Chinese PKU patients was analyzed. Then 20 variants (p.L52F, p.R86P, p.L128P, p.L142P, p.D163N, p.C203G, p.E214G, p.F260L, p.M276T, p.L311R, p.P314A, p.L364F, p.Q375H, p.F382I, p.A395S, p.V412D, p.E108*, p.C203*, p.C284* and p.E353*) were expressed in COS-7 cells. The residual activities and protein expression levels were detected by isotope-dilution liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) and Western blotting, respectively. We compared the results of the phenotypic prediction based on APV and PAH activity respectively, and further explored the relationship between residual activity and phenotype in PKU patients.We reported 9 newly discovered PAH variants for the first time, thereby expanding the spectrum of PAH variants. Among the 20 variants in our assay, 8 variants showed mild impaired residual activities (48–92%) and approximately normal protein expression levels compared to the wild-type PAH. In contrast, 9 variants showed severely impaired residual activities (0–34%) and reduced protein expression. However, three variants (p.L52F, p.F260L and p.P314A) showed impaired residual activities (5%, 32% and 29%), although the proteins were well expressed. We assigned APV scores for 14 variants, in which the results of the phenotypic prediction were consistent for 12/14 (86%) variants based on APV and residual activity respectively, and the residual activity correctly predicted 17/22 (77%) of the patients. Our study helped to further understand the genotype-phenotype correlation in PKU patients.

A strategy for identification and structural characterization of oplopane- and bisabolane-type sesquiterpenoids from Tussilago farfara L. by multiple scan modes of mass spectrometry

A liquid chromatography–electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS)-based dereplicative method was developed for identifying oplopane- and bisabolane-type sesquiterpenoids from buds of Tussilago farfara L. The analysis of these chemical analogues, sesquiterpene esters, is challenging by MS-based nontargeted metabolomic approaches because of their in-source fragmentation and structural diversity. To profile these sesquiterpenoids, four diagnostic ions (m/z 215.143, 217.158, 229.123, and 231.138) were suggested in the positive ion mode and the developed method utilized two sequential MS/MS scan modes to identify common skeletons and investigate the fragmentation patterns of their parent molecules. Precursor ion scan by triple quadrupole MS/MS provided the parent molecular ions from their diagnostic ions, and product ion scan by quadrupole time-of-flight MS/MS confirmed their fragmentation behaviors. Under the optimized UHPLC-MS/MS method, 74 sesquiterpenoids were characterized from the Farfarae Flos and 11 compounds were isolated for the method validation. Among those compounds, three sesquiterpenoids were newly separated from the Farfarae Flos. Furthermore, the diagnostic ions and the MS/MS fragment behaviors were applied to the accurate quantification of the 8 isolated sesquiterpenoids. Therefore, the developed LC–MS/MS-based method highlighted the chemical composition of the Farfarae Flos and could be extended to the screening and quantification of other sesquiterpene esters.