Qualitative and quantitative determination of major saponins in Paris and Trillium by HPLC-ELSD and HPLC–MS/MS

High-performance liquid chromatographic (HPLC) with evaporative light scattering detection (ELSD) and HPLC with electrospray ionization multistage tandem mass spectrometry (HPLC–ESI-MSⁿ) were used to identify and quantify steroid saponins in Paris and Trillium plants. The content of the known saponins such as Paris I, II, III, V, VI, VII, H, gracillin and protodioscin in Paris and Trillium plants was determined simultaneously using the developed HPLC-ELSD method. Furthermore, other 12 steroid saponins were identified by HPLC–ESI(+/−)-MSⁿ detection. In the end, a developed analytical procedure was proved to be a reliable and rapid method for the quality control of Paris and Trillium plants. In addition, the alternative resources for Paris yunnanensis used as a traditional Chinese medicine were discovered according to the hierarchical clustering analysis of the saponin fraction of these plants.     

LC/MS/MS method for analysis of E₂ series prostaglandins and isoprostanes

15-series prostaglandins (PGE₂s) and isoprostanes (isoPGE₂s) are robust biomarkers of oxidative stress, possess potent biological activity, and may be derived through cyclooxygenase or free radical pathways. Thus, their quantification is critical in understanding many biological processes where PG, isoPG, or oxidative stress are involved. LC/MS/MS methods allow a highly selective, sensitive, simultaneous analysis for prostanoids without derivatization. However, the LC/MS/MS methods currently used do not allow for simultaneous separation of the major brain PGE₂/D₂ and isoPGE₂ without derivatization and multiple HPLC separations. The developed LC/MS/MS method allows for the major brain PGE₂/PGD₂/isoPGE₂ such as PGE₂, entPGE₂, 8-isoPGE₂, 11β-PGE₂, PGD₂, and 15(R)-PGD₂ to be separated and quantified without derivatization. The method was validated by analyzing free and esterified isoPGE₂ in mouse brains fixed with head-focused microwave irradiation before or after global ischemia. Using the developed method, we report for the first time the esterified isoPGE₂ levels in brain tissue under basal conditions and upon global ischemia and demonstrate a nonreleasable pool of esterified isoPG upon ischemia. In addition, we demonstrated that PGE₂s found esterified in the sn-2 position in phospholipids are derived from a free radical nonenzymatic pathway under basal conditions. Our method for brain PG analysis provides a high level of selectivity to detect changes in brain PG and isoPG mass under both basal and pathological conditions.     

Simultaneous quantification of the organophosphorus pesticides dimethoate and omethoate in porcine plasma and urine by LC–ESI-MS/MS and flow-injection-ESI-MS/MS

Dimethoate is an organophosphorus toxicant used in agri- and horticulture as a systemic broad-spectrum insecticide. It also exhibits toxic activity towards mammalian organism provoked by catalytic desulfuration in the liver producing its oxon-derivative omethoate thus inhibiting acetylcholinesterase, initiating cholinergic crisis and ultimately leading to death by respiratory paralysis and cardiovascular collapse. Pharmaco- and toxicokinetic studies in animal models help to broaden basic understanding of medical intervention by antidotes and supportive care. Therefore, we developed and validated a LC–ESI-MS/MS method suitable for the simultaneous, selective, precise (RSD_intra-day 1–8%; RSD_inter-day 5–14%), accurate (intra-day: 95–107%; inter-day: 90–115%), and robust quantification of both pesticides from porcine urine and plasma after deproteinization by precipitation and extensive dilution (1:11,250 for plasma and 1:40,000 for urine). Accordingly, lower limits of quantification (0.24–0.49 μg/ml plasma and 0.78–1.56 μg/ml urine) and lower limits of detection (0.12–0.24 μg/ml plasma and 0.39–0.78 μg/ml urine) were equivalent to quite low absolute on-column amounts (1.1–2.1 pg for plasma and 2.0–3.9 pg for urine). The calibration range (0.24–250 μg/ml plasma and 0.78–200 μg/ml urine) was subdivided into two linear ranges (r² ≥ 0.998) each covering nearly two orders of magnitude. The lack of any interfering peak in 6 individual blank specimens from plasma and urine demonstrated the high selectivity of the method. Furthermore, extensive sample dilution causing lowest concentration of potentially interfering matrix ingredients prompted us to develop and validate an additional flow-injection method (FI-ESI-MS/MS). Validation characteristics were as good as for the chromatographic method but sample throughput was enhanced by a factor of 6. Effects on ionization provoked by plasma and urine matrix from 6 individuals as well as in the presence of therapeutics (antidotes) administered in an animal study were investigated systematically underling in the reliability of the presented methods. Both methods were applied to porcine samples derived from an in vivo animal study.     

A fast and sensitive HPLC–MS/MS analysis and preliminary pharmacokinetic characterization of ergone in rats

A fast and sensitive HPLC–APCI-MS/MS method was developed for the determination of ergosta-4,6,8(14),22-tetraen-3-one (ergone) in rat plasma. The plasma sample containing ergone and ergosterol (internal standard) were simply treated with acetone to precipitate and remove proteins and the isolated supernatants were directly injected into the HPLC–APCI-MS/MS system. Chromatographic separation was performed on a 1.8 μm Zorbax SB-C18 column (100 mm × 3.0 mm) with a 97:3 (v/v) mixed solution of methanol and 0.1% aqueous formic acid being used as mobile phase. Quantification was performed by multiple selected reactions monitoring (MRM) of the transitions with (m/z)⁺ 393–268 for ergone and (m/z)⁺ 379–69 for the IS. The method was validated in the concentration range of 5–1600 ng/mL for ergone. The precision of the assay (RSD%) was less than 10.5% at all concentrations levels within the tested range and adequate accuracy, and the limit of detection was 1.5 ng/mL. The absolute recoveries of both ergone and ergosterol from the plasma were more than 95%. The developed method has been successfully applied to the pharmacokinetic study of the drug in SD rats.     

Sub-proteomic fractionation,iTRAQ, and OFFGEL-LC–MS/MS approaches to cardiac proteomics

Using an in solution based approach with a sub-proteomic fraction enriched in cardiac sarcomeric proteins; we identified protein abundance in ischemic and non-ischemic regions of rat hearts stressed by acute myocardial ischemia by ligating the left-anterior descending coronary artery in vivo for 1 h without reperfusion. Sub-cellular fractionation permitted more in depth analysis of the proteome by reducing the sample complexity. A series of differential centrifugations produced nuclear, mitochondrial, cytoplasmic, microsomal, and sarcomeric enriched fractions of ischemic and non-ischemic tissues. The sarcomeric enriched fractions were labeled with isobaric tags for relative quantitation (iTRAQ), and then fractionated with an Agilent 3100 OFFGEL fractionator. The OFFGEL fractions were run on a Dionex U-3000 nano LC coupled to a ThermoFinnigan LTQ running in PQD (pulsed Q dissociation) mode. The peptides were analyzed using two search engines MASCOT (MatrixScience), and MassMatrix with false discovery rate of < 5%. Compared to no fractionation prior to LC–MS/MS, fractionation with OFFGEL improved the identification of proteins approximately four-fold. We found that approximately 22 unique proteins in the sarcomeric enriched fraction had changed at least 20%. Our workflow provides an approach for discovery of unique biomarkers or changes in the protein profile of tissue in disorders of the heart.     

LC–MS/MS method development and validation for the determination of polymyxins and vancomycin in rat plasma

Simple, sensitive and robust liquid chromatography–tandem mass spectrometer (LC–MS/MS) methods were developed and validated for the determination of lipopeptide polymyxins and glycopeptide vancomycin in rat plasma. The effect of trichloroacetic acid (TCA) concentration on sample recoveries (peak area of sample recovered from plasma/peak area of sample from neat solvent solutions) was studied and an optimized concentration of 30% TCA were determined that gives the best sample recovery for the peptides from rat plasma. The effect of the TCA concentration on the chromatographic behavior of peptides was studied on a Phenomenex Jupiter C18 5 μ 300 Å 50 mm × 2 mm column using a mobile phase with a pH of 2.8. Other than protein precipitation, TCA also acted as ion pairing reagent and was only present in the samples but not in the mobile phases. The data demonstrated that by increasing the TCA concentration, the analyte retention and sensitivity were improved. The absence of TCA in mobile phase helped to reduce the ion source contamination and to achieve good reproducibility. The plasma method was linearly calibrated from 5 to 5000 ng/mL for polymyxins with precisions to be of 2.3–10.8%, and accuracies to be 91.7–107.4% for polymyxin B1, B2, E1, E2, respectively. For vancomycin the calibration is from 1 to 5000 ng/mL with precisions to be of 7.8–10.3 and accuracies to be 96.2–102.0%. The LLOQs corresponding with a coefficient of variation less than 20% were 7.5, 18.1, 7.3, 5.0 and 1.0 ng/mL for polymyxin B1, B2, E1, E2 and vancomycin, respectively.     

Efficient analysis and extraction of MS/MS result data from Mascot™ result files

Background  

Mascot™ is a commonly used protein identification program for MS as well as for tandem MS data. When analyzing huge shotgun proteomics datasets with Mascot™'s native tools, limits of computing resources are easily reached. Up to now no application has been available as open source that is capable of converting the full content of Mascot™ result files from the original MIME format into a database-compatible tabular format, allowing direct import into database management systems and efficient handling of huge datasets analyzed by Mascot™.     

GC–MS and GC–MS/MS measurement of the cardiovascular risk factor homoarginine in biological samples

l-Homoarginine (hArg) has recently emerged as a novel cardiovascular risk factor and to herald a poor prognosis in heart failure patients. Here, we report on the development and thorough validation of gas chromatography–mass spectrometry (GC–MS) and gas chromatography–tandem mass spectrometry (GC–MS/MS) methods for the quantitative determination of hArg in biological samples, including human plasma, urine and sputum. For plasma and serum samples, ultrafiltrate (10 µL; cutoff, 10 kDa) was used. For urine samples, native urine (10 µL) was used. For sputum, protein precipitation by acetone was performed. hArg is derivatized to its methyl ester tri(N-pentafluoropropionyl) derivative; de novo synthesized trideutero-methyl ester hArg is used as the internal standard (IS). Alternatively, [guanidino-¹⁵N₂]-arginine can be used as an IS. Quantitative analyses were performed after electron-capture negative-ion chemical ionization by selected-ion monitoring in GC–MS and selected-reaction monitoring in GC–MS/MS. We obtained very similar hArg concentrations by GC–MS and GC–MS/MS, suggesting that GC–MS suffices for accurate and precise quantification of hArg in biological samples. In plasma and serum samples of the same subjects very close hArg concentrations were measured. The plasma-to-serum hArg concentration ratio was determined to be 1.12 ± 0.21 (RSD, 19 %), suggesting that blood anticoagulation is not a major preanalytical concern in hArg analysis. In healthy subjects, the creatinine-corrected urinary excretion of hArg varies considerably (0.18 ± 0.22 µmol/mmol, mean ± SD, n = 19) unlike asymmetric dimethylarginine (ADMA, 2.89 ± 0.89 µmol/mmol). In urine, hArg correlated with ADMA (r = 0.475, P = 0.040); in average, subjects excreted in the urine about 17.5 times more ADMA than hArg. In plasma of healthy humans, the concentration of hArg is of the order of 2 µM. hArg may be a low-abundance constituent of human plasma proteins. The GC–MS and GC-MS/MS methods we report in this article are useful to study the physiology and pathology of hArg in experimental and clinical settings.     

HPLC-DAD和LC/MS/MS测定豆奶中三聚氰胺

目的：建立二极管阵列高效液相色谱仪和三重四级杆液质联用仪对豆奶中三聚氰胺的测定方法。方法：采用三氯乙酸和乙腈为提取剂、蛋白质为沉淀剂,提取液过净化柱纯化。结果：三重四级杆液质联用法对三聚氰胺的检出限为0-001 5 mg/kg,标准曲线在0-01~0-5 μg/mL范围内,R2为0-999 8,线性良好,再回收率为85 %~89 %,适用于检测低浓度的样品;二极管阵列高效液相色谱法检出限为0-024 mg/kg,标准曲线在0-5~100 μg/mL范围内,R2为0-999 9,线性良好,回收率为83 %~91 %,可以快速地对高浓度样品进行筛查。结论以上两种检测方法结合使用,可检测0-01~100 mg/kg的三聚氰胺含量,极大地拓宽了检测范围。     

Quantification of isradipine in human plasma using LC–MS/MS for pharmacokinetic and bioequivalence study

A highly sensitive and rapid method for the analysis of isradipine in human plasma using liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) was developed. The procedure involves a simple liquid–liquid extraction of isradipine and amlodipine (IS, internal standard) with methyl-t-butyl ether after alkaline treatment and separation by RP-HPLC. Detection was performed by positive ion electrospray ionization (ESI) in multiple reaction monitoring (MRM) mode, monitoring the transitions m/z 372.1 → m/z 312.2 and m/z 408.8 → m/z 237.9, for quantification of isradipine and IS, respectively. The standard calibration curves showed good linearity within the range of 10 to 5000 pg/mL (r² ≥ 0.9998). The lower limit of quantitation (LLOQ) was 10 pg/mL. The retention times of isradipine (0.81 min) and IS (0.65 min) suggested the potential for high throughput of the proposed method. In addition, no significant metabolic compounds were found to interfere with the analysis. This method offered good precision and accuracy and was successfully applied for the pharmacokinetic and bioequivalence studies of 5 mg of sustained-release isradipine in 24 healthy Korean volunteers.     

Development,validation and comparison of LC–MS/MS and RIA methods for quantification of vertebrates-like sex-steroids in prosobranch molluscs

The role of vertebrate-like sex-steroids (testosterone, T, progesterone, P, and 17β-estradiol, E2) in molluscs is still debated, but they could represent potential biomarkers of endocrine disruption. A radioimmunoassay (RIA) and a liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) methods have been developed and compared to measure their levels in a gastropod snail Potamopyrgus antipodarum. Both methods showed a good reproducibility despite the complex matrix and the very low levels of vertebrate-like sex-steroids. Only T and P were detected using the LC–MS/MS method, while the RIA method reached lower detection limits and enabled the detection of all three steroids. Results indicated that T and P were mainly present as unconjugated forms. Both methods were compared in the analysis of snails exposed to waste water treatment plant effluents and led to the same conclusions concerning the modulation of steroids levels. Moreover, they both were in agreement concerning T measurements. On the other hand, LC–MS/MS appeared to be more suitable when analyzing P levels due to a low sensitivity of the RIA method. As E2 was not measured using the LC–MS/MS method because of a higher detection limit compared to the other steroids, the results obtained with the RIA method should be interpreted with caution. LC–MS/MS remains the gold standard for sex-steroid determinations, however a relevant and alternative method based on RIA was developed, requiring fewer organisms. RIA seems a promising method as a screening tool for experimental use, allowing comparison of sex-steroid levels in the mudsnail both in laboratory and in field experiments.     

Metabolomic and elemental analysis of camel and bovine urine by GC–MS and ICP–MS

Recent studies from the author’s laboratory indicated that camel urine possesses antiplatelet activity and anti-cancer activity which is not present in bovine urine. The objective of this study is to compare the volatile and elemental components of bovine and camel urine using GC–MS and ICP–MS analysis. We are interested to know the component that performs these biological activities. The freeze dried urine was dissolved in dichloromethane and then derivatization process followed by using BSTFA for GC–MS analysis. Thirty different compounds were analyzed by the derivatization process in full scan mode. For ICP–MS analysis twenty eight important elements were analyzed in both bovine and camel urine. The results of GC–MS and ICP–MS analysis showed marked difference in the urinary metabolites. GC–MS evaluation of camel urine finds a lot of products of metabolism like benzene propanoic acid derivatives, fatty acid derivatives, amino acid derivatives, sugars, prostaglandins and canavanine. Several research reports reveal the metabolomics studies on camel urine but none of them completely reported the pharmacology related metabolomics. The present data of GC–MS suggest and support the previous studies and activities related to camel urine.     

Online immunoaffinity LC/MS/MS. A general method to increase sensitivity and specificity: How do you do it and what do you need?

There is an increased emphasis on hyphenated techniques such as immunoaffinity LC/MS/MS (IA-LC/MS/MS) or IA-LC/MRM. These techniques offer competitive advantages with respect to sensitivity and selectivity over traditional LC/MS and are complementary to ligand binding assays (LBA) or ELISA's. However, these techniques are not entirely straightforward and there are several tips and tricks to routine sample analysis. We describe here our methods and procedures for how to perform online IA-LC/MS/MS including a detailed protocol for the preparation of antibody (Ab) enrichment columns. We have included sample trapping and Ab methods. Furthermore, we highlight tips, tricks, minimal and optimal approaches. This technology has been shown to be viable for several applications, species and fluids from small molecules to proteins and biomarkers to PK assays.     

Simultaneous determination of a novel antifibrotic agent and three metabolites in human urine by LC–MS/MS

A robust and validated high performance liquid chromatography tandem mass spectrometry (LC–MS/MS) method has been developed for simultaneous determination of F351 (5-methyl-1-(4-hydroxylphenyl)-2-(1H)-pyridone) and three major metabolites in human urine sample. This assay method has also been validated in terms of selectivity, linearity, lower limit of quantification (LLOQ), accuracy, precision, stability, matrix effect and recovery. Chromatography was carried out on an XTerra RP 18 column and mass spectrometric analysis was performed using an API 4000 mass spectrometer coupled with electro-spray ionization (ESI) source in the positive ion mode. The MRM transitions of m/z 202 → 109, 232 → 93, 282 → 202 and 378 → 202 were used to quantify F351 and three metabolites, respectively. Retention times for F351 and three metabolites were 2.54, 1.38, 1.53 and 1.34 min, respectively. The assay was validated from 20 to 4000 ng/mL for F351 and M1, from 80 to16,000 ng/mL for M2 and M3. Intra- and inter-day precision for all analytes was <6.3%, method accuracy was between −11.2 and 0.3%. This assay was used to support a clinical study where multiple oral doses were administered to healthy subjects to investigate the pharmacokinetics, safety, and tolerability of F351.     

Simple,sensitive and rapid HPLC–MS/MS method for the determination of cepharanthine in human plasma

A rapid, sensitive and specific method for the determination of cepharanthine in human plasma using high performance liquid chromatography coupled with tandem mass spectrometry (HPLC–MS/MS) was described. Cepharanthine and the internal standard (I.S.), telmisartan, were extracted from human plasma by methanol to precipitate the protein. A centrifuged upper layer was then evaporated and reconstituted with 100 μL methanol. Chromatographic separation was performed on an AGILENT XDB-C₈ column (150 mm × 2.1 mm, 5.0 μm, Agilent, USA) using a gradient mobile phase with 1 mmol/L ammonium acetate in water with 0.05% formic acid and methanol. Detection and quantitation was performed by MS/MS using electrospray ionization (ESI) and multiple reaction monitoring (MRM) in the positive ion mode. The most intense [M+H]⁺ MRM transition of cepharanthine at m/z 607.3 → 365.3 was used for quantitation and the transition at m/z 515.5 → 276.4 was used to monitor telmisartan. The calibration curve was linear within the concentration range of 0.5–200.0 ng/mL (r = 0.9994). The limit of quantification (LOQ) was 0.5 ng/mL. The extraction recovery was above 81.1%. The accuracy was higher than 92.3%. The intra- and inter-day precisions were less than 9.66%. The method was accurate, sensitive and simple and was successfully applied to a pharmacokinetic study after single intravenous administration of 50 mg cepharanthine in 12 healthy Chinese volunteers.     

LC–MS/MS characterization of combined glycogenin-1 and glycogenin-2 enzymatic activities reveals their self-glucosylation preferences

Glycogen synthesis is initiated by self-glucosylation of the glycosyltransferases glycogenin-1 and -2 that, in the presence of UDP-glucose, form both the first glucose-O-tyrosine linkage, and then stepwise add a series of α1,4-linked glucoses to a growing chain of variable length. Glycogen-1 and -2 coexist in liver glycogen preparations where the proteins are known to form homodimers, and they also have been shown to interact with each other. In order to study how glycogenin-1 and -2 interactions may influence each other's glucosylations we setup a cell-free expression system for in vitro production and glucosylation of glycogenin-1 and -2 in various combinations, and used a mass spectrometry based workflow for the characterization and quantitation of tryptic glycopeptides originating from glycogenin-1 and -2. The analysis revealed that the self-glucosylation endpoint was the incorporation of 4–8 glucose units on Tyr 195 of glycogenin-1, but only 0–4 glucose units on Tyr-228 of glycogenin-2. The glucosylation of glycogenin-2 was enhanced to 2–4 glucose units by the co-presence of enzymatically active glycogenin-1. Glycogenin-2 was, however, unable to glucosylate inactive glycogenin-1, at least not an enzymatically inactivated Thr83Met glycogenin-1 mutant, recently identified in a patient with severe glycogen depletion.