Simple LC-MS Research Articles

Bioanalytical Method Development and Validation of MEM-PLGANanoparticles via Nasal Route Administration.

The aim of this effort is to increase extended pharmaceutical release by systematizing PN elaboration for ME, a novel treatment for Alzheimer’s disease. Bioanalysis is an essential part in drug discovery and development. Bioanalysis is related to the analysis of analytes (drugs, metabolites, biomarkers) in biological samples and it involves several steps from sample collection to sample analysis and data reporting. A simple, sensitive, and quick LC-MS technique for quantifying MEM in rat plasma was developed and validated. Chromatography was carried out on a C18 column (4.6 × 75mm, 3.5μm). ME and its internal standard, NLB, were extracted by direct protein precipitation and were chosen as the sample treatment method rather than liquid-liquid extraction techniques and evaluated with LC-MS/MS in multiple-reaction monitoring (MRM) mode. This approach demonstrated intra- and interday precision in the ranges of 2.1-3.7 and 1.4-7.8%. Furthermore, intra- and interday accuracy ranged from 95.6 to 99.8, and 95.7-99.1%, respectively. MEM and NLB showed mean recovery rates of 86.07 ± 6.87 and 80.31 ± 5.70%, respectively. The stated method was effectively applied in the bioequivalence study of MEM and evaluated its potential for treating Alzheimer’s disease via the nasal route. MEM administered via the nasal route will demonstrate improved bioavailability and efficacy in the treatment of Alzheimer’s disease compared to current therapies.

Development and Validation of Simple and Stable LCMS Method for the Quantification of Potential Genotoxic Impurities in Ozenoxacin Pure Drug and its Commercial Preparations

Ozenoxacin is an antibiotic drug prescribed to treat various skin infections caused by various bacteria. Various chemical mechanisms such as stille coupling, Buchwald–Hartwig coupling, cyclization and saponification are involved during the process of synthesis of Ozenoxacin. In the process of synthesis, there is a possibility of the formation of related impurities and among them, some are genotoxic impurities. To date, in literature, there is no method reported for analysing Potential Genotoxic Impurities (PGIs) in Ozenoxacin and hence this study was initiated to develop an LCMS method for quantification of two genotoxic impurities of Ozenoxacin viz., nitroso impurity, ester impurity. The analytes were resolved on Alltima C18 column (150×4.6mm; 5 μm particle size) using 0.01 mM ammonium acetate at pH 4.8 and methanol in 80:20 (v/v) at 0.5 mL/min flow rate and 10 μ sample injection volume. The multiple reaction monitoring of the mass fragments confirms the parent ion at m/z of 364, 393 and 496 for Ozenoxacin, Nitroso and Ester impurity respectively with characteristic product ion at m/z 196. The method has a linearity range of 0.05 μg/mL to 1.0 μg/mL for three analytes with detection limits of 0.015, 0.011 and 0.015 μg/mL for Ester impurity, Ozenoxacin and Nitroso impurity respectively. The method was validated and produces acceptable results and can successfully separate the potential genotoxic impurities in spiked commercial samples. Based on the findings, it was concluded that this method can be practically useful for the identification and quantification of potential genotoxic impurities and may apply to the safe use of Ozenoxacin in clinical treatment.

Development and validation of a bioanalytical method for the quantification of methotrexate from serum and capillary blood using volumetric absorptive microsampling (VAMS) and on-line solid phase extraction (SPE) LC-MS

High-dose methotrexate is part of the polychemotherapy protocols for the treatment of Acute lymphoblastic leukaemia (ALL) with therapeutic drug monitoring (TDM) to adjust leucovorin rescue. An immunoassay is commonly used to analyse serum samples collected via venous blood sampling. However, immunoassays cannot distinguish between the parent drug and its metabolites. Besides, the blood volume required by venous blood sampling is high. Therefore, the aim of this project was to develop a fast, simple, reliable and cost-efficient micro sampling bioanalytical method using capillary blood to minimize the harm of children and to analyse both methotrexate and its metabolites. To achieve this aim, a LC-MS method with on-line solid phase extraction (SPE) for the simultaneous detection of methotrexate and its metabolites from capillary blood using volumetric-absorptive-microsampling (VAMS) technology was developed and fully validated. Besides, the method was also validated and modified for serum samples to compare the results with the immunoassay. A single-quadrupole MS detector was used for detection. Through the use of on-line SPE technology, a lower limit of quantitation of 0.03 µM for MTX and 7-OH-MTX and of 0.05 µM for DAMPA from a 10 μL capillary blood sample was achieved. The accuracy is between 90.0 and 104% and the precision between 4.7 and 12% for methotrexate and its metabolites, respectively. Because of the cross reactivity of the immunoassay a cross-validation was not successful. Besides, a correlation factor of 0.46 for MTX between plasma and whole-blood was found.A fast, simple, reliable and cost-efficient extraction and analysis LC-MS method could be developed and validated, which is applicable in ambulatory and clinical care.

Development and validation of a chiral LC-MS method for the enantiomeric resolution of (+) and (-)-Mebeverine Hydrochloride in bulk drug by using polysaccharide-based chiral stationary phase

The aim of this study was to develop a simple, specific, sensitive and precise LC-MS method for the determination of mebeverine enantiomers in pharmaceutical formulations and it was validated as per ICH guidelines. The chromatographic separation was achieved on Phenomenex® Lux Cellulose 1 (250 mm x 4.6 mm i.d, 5μm particle size) column using mobile phase system containing acetonitrile: 10 mM ammonium acetate (85:15) at the flow rate of 0.8 mL/min. In the spectral investigation by LC-MS, the standard solution of Mebeverine showed major peak at m/z of 430.70, which was assigned to the [M+H] ion of Mebeverine. The described method was linear over the range of 15–75 ng /mL for (±) Mebeverine enantiomers with a correlation coefficient (r2 = 0.999). Detection limits and quantification limits of (+) Mebeverine and (-) Mebeverine were found to be 5 ng/mL and 15 ng/mL respectively. The recovery study of mebeverine from tablet formulation was found to be 99.16%. Mebeverine standard solution and mobile phase were found to be stable for at least 48h. The Mebeverine enantiomers were well resolved with mean retention times of about 1.16 min and 1.20 min respectively. The developed method was extensively validated and proved to be robust, accurate, precise, and suitable for analysis of Mebeverine enantiomers in tablet dosage form.

Development of a SPE-LC-MS Method for the Quantitation of Palbociclib and Abemaciclib in Human Plasma

Palbociclib and abemaciclib are two cyclin-dependent kinases 4 and 6 used for breast cancer treatment. Levels of these medicines present a significant interindividual variability, so monitoring those concentrations might be necessary in therapy. Most of the methods presented so far in the literature use simple protein precipitation of plasma proteins as sample preparation method followed by direct injection of the supernatant into the LC instrument, preceded or not by a simple filtration step. Within that approach, the probability of injecting proteins in the chromatographic system is increased. With the purpose of obtaining a cleaner extract of the drugs, we developed and validated a simple and accurate LC-MS method for determining palbociclib and abemaciclib in human plasma. Solid phase extraction (SPE) using Oasis PRiME HLB® cartridges was used for plasma sample preparation. The method provided clean extracts with a recovery extraction higher than 85% for both compounds. Separation was achieved by high-performance liquid chromatography (HPLC), using a C18 (4.6 × 50 mm) column, with a gradient elution of ammonium acetate/acetic acid-acetonitrile as the mobile phase. Detection was performed by mass spectrometry (MS) in single ion recording (SIR) mode. Intra-day and inter-day precision data for both analytes were 3.8-7.2% and 3.6-7.4%, respectively. Calibration curves were both linear between 2 and 400 ng/mL with a correlation coefficient higher than 0.998. The LC-MS method can be used to quantify the drugs in human plasma in routine analysis. The method proved to be useful in determining real plasma levels in patients involved in cancer therapy. Drug concentrations were determined in a 10 min run-time, including re-equilibration of the column.

Development and validation of a bioanalytical method for the quantification of axitinib from plasma and capillary blood using volumetric absorptive microsampling (VAMS) and on-line solid phase extraction (SPE) LC-MS

For therapeutic drug monitoring (TDM) of axitinib, the new volumetric absorption microsampling technology (VAMS™) was applied to obtain capillary blood samples in an ambulatory setting and the results were compared to plasma samples as the gold standard. On-line solid phase extraction (SPE) applying a Turboflow HTLC Cyclone™ 1.0 × 500 mm column was used to reduce costs and working time. For the analytical separation, a Kinetex 2.6 µm C18 100 Å, 100 × 3.0 mm column with a flow rate of 0.3 mL/min in gradient mode was utilised. The mobile phase consisted of acetonitrile, water and formic acid (A: 05:95:0.1 v/v and B: 95:05:0.1 v/v). For the detection, a single-quadrupole MS detector was used. Through the use of on-line SPE technology, it is possible to reach a LLOQ of 0.5 µg/L from a 10 µL capillary blood sample. For lower concentrations, a MS/MS-detector coupled with the same chromatographic system was applied reaching a LLOQ of 0.04 µg/L. This newly developed method was validated with both detectors at different calibration ranges for plasma and capillary blood as matrix. The precision of the within- and between-runs was within a range of 0.6–7.8% and 1.8 – 14% CV, respectively, while the accuracy was within a range of 81.2–115% and 87.7–116%, respectively. A reliable, simple, less personnel-intensive and cost-efficient extraction and analysis LC-MS and LC-MS/MS method could be developed and validated, which is applicable in ambulatory and clinical care.

Volumetric Absorptive Microsampling as a New Biosampling Tool for Monitoring of Tamoxifen, Endoxifen, 4-OH Tamoxifen and N-Desmethyltamoxifen in Breast Cancer Patients.

IntroductionIn this research, we used a volumetric absorptive microsampling (VAMS) technique to collect blood samples from the patients. A rapid and simple sample preparation method and LC-MS.MS assay was then developed and validated for the simultaneous analysis of tamoxifen and its three active metabolites.MethodsVAMS extraction was performed in methanol by sonication-assisted extraction method for 25 min after 2 hof VAMS drying. Separation was carried out using Acquity UPLC BEH C18 column (2.1 x 100 mm; 1.7 µm), with a flow rate of 0.2 mL/min, and the mobile phase gradient of formic acid 0.1% and formic acid 0.1% in acetonitrile for 5 min. The multiple reaction monitoring (MRM) values were set at m/z 358.31>58.27 for N-desmethyltamoxifen, m/z 372.33>72.28 for tamoxifen, m/z 388.22>72.28 for 4-hydroxytamoxifen, m/z 374.25>58.25 for endoxifen, and m/z 260.26>116.12 for propranolol.Results and DiscussionThe lower limit of quantification value (LLOQ) was 2.50 ng/mL for tamoxifen, 2.50 ng/mL for endoxifen, 1.50 ng/mL for 4-hydroxitamoxifen, and 2.00 ng/mL for N-desmethyltamoxifen. Accuracy (%bias) and precision (%CV) were within 20% for LLOQ and 15% for other concentrations. There were no interference responses >20% of the LLOQ and 5% of the internal standard. The level of ion suppression in all analytes was less than 7%. The preparation system developed in this study successfully extracted more than 90% of analytes from the matrix with precision below 15%. Carryover was shown to be below 6% in all analytes. Stability of analytes in VAMS was demonstrated for up to 30 days, under room temperature storage in a sealed plastic bag with desiccant. This method was successfully applied to analyze tamoxifen and the metabolites level in 30 ER+ breast cancer patients.

Development and validation of a sensitive, fast and simple LC-MS / MS method for the quantitation of favipiravir in human serum

Favipiravir is a broad-spectrum inhibitor of viral RNA polymerase. It is currently used as a possible treatment for coronavirus disease 2019 (COVID-19). Pre-clinical or clinical trials of favipiravir require robust, sensitive, and accurate bioanalytical methods for quantitation of favipiravir levels. Recently, several studies have been reported about developing a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for measuring favipiravir levels. However, these methods were validated predominantly for plasma samples, electrospray ionization was operated only in negative or positive mode, and clinical application of these methods has not been applied for patients with COVID-19. This study aimed was to develop a validated LC-MS/MS method for the measurement of favipiravir levels in positive and negative electrospray ionization mode and to perform a pilot study in patients with COVID-19 receiving favipiravir to demonstrate the applicability of this method in biological samples. Simple protein precipitation was used for the extraction of favipiravir from the desired matrix. Favipiravir levels were quantitated using MS / MS with an electrospray ionization source in positive and negative multiple reaction monitoring (MRM) mode. The chromatographic detection was performed on a reverse-phase Phenomenex C18 column (50 mm × 4.6 mm, 5 µm, 100 Å) with gradient elution using 0.1% formic acid in water and 0.1% formic acid in methanol as mobile phase. The method was linear over the concentration ranges of 0.048–50 µg/mL (in negative ionization mode) and 0.062–50 µg/mL (in positive ionization mode) with a correlation coefficient (r2) better than 0.998. The total run time was 3.5 min. The intra-assay and inter-assay %CV values were less than 7.2% and 8.0%, respectively. A simple, rapid and robust LC-MS / MS method was developed for the measurement of favipiravir and validation studies were performed. The validated method was successfully applied for drug level measurement in COVID-19 patients receiving favipiravir.

Simultaneous determination the concentration change of ketoconazole and dexamethasone acetate: application to drug-drug interaction in human keratinocyte

BackgroundThe combination of ketoconazole and dexamethasone acetate is one of the commonly used treatments in dermatology regiment for skin inflammation accompanied by fungal infections. This interaction of ketoconazole and dexamethasone may make the drug much effective, decrease the adverse reaction or toxic effects. At present, there was no information about the interaction of these two external drugs. Therefore, it was necessary to build a model to measure the levels and evaluate the interaction of ketoconazole and dexamethasone acetate in skin cells. MethodsIn our study, the determination methodology of ketoconazole and dexamethasone acetate in human keratinocyte (HaCaT cells) was established and the interaction of these two drugs in cells was explored. HaCaT cells were cultured in medium containing ketoconazole, then they were sequentially cultured with or without dexamethasone acetate treatment for another 1, 2, 4, 8 and 12 h. The samples were then harvested and the concentrations were quantified by enhanced BCA (the bicinchoninic acid) protein assay. Furthermore, the analytes in the cell suspension were also prepared and analyzed by LC-MS method. ResultsAs a result, ketoconazole and dexamethasone acetate were detected at the concentration range of 0.02 to 5 μg/mL and 0.2 to 100 μg/mL, respectively. The RSD (relative standard deviation) and RE (relative error) of precision and accuracy of the two analytes in the cell suspension were all less than 15 %. The matrix effect values variations were all less than 15%. The results showed that there was no significant difference in the concentration of ketoconazole with or without dexamethasone acetate treatment within 12 h. ConclusionsA simple, sensitive and rapid LC-MS method which could quantify these two analytes in cells simultaneously was developed for the first time. The results of the concentration changes meant that no interaction occurred when dexamethasone sequentially administrated for 12 h after ketoconazole treatment. This method was successfully applied to establish the cell pharmacokinetics methodology and preliminary studied the metabolism of drug-drug interaction of ketoconazole and dexamethasone acetate.

Simple and Rapid HPLC Separation and Quantification of Flavonoid, Flavonolignans, and 2,3-Dehydroflavonolignans in Silymarin.

Herbal preparations from Silybum marianum have been used since the fourth century BC in liver disease treatment and against numerous other pathologies. Consumption of silymarin containing drugs and food supplements continues to increase. Precise, fast, reliable, and complex determination of all components of silymarin preparations is paramount for assessing its pharmacological quality. We present here simple and fast HPLC-DAD and LC-MS analytical methods for the determination and quantification of all known silymarin components, including 2,3-dehydroflavonolignans that has not been achieved so far. The first method, using a common C18 column, allows baseline separation of previously inseparable silychristin A, B, isosilychristin, and silydianin. Moreover, this method allowed detection of three so far unknown silymarin components. In addition, the first analytical separation of enantiomers of 2,3-dehydrosilybin was achieved using a Lux 3μ Cellulose-4 chiral column, providing even more accurate description of silymarin composition. 2,3-Dehydroflavonolignans were isolated for the first time from silymarin using preparative chromatography on C18 and ASAHIPAK columns, and 2,3-dehydrosilychristin and 2,3-dehydrosilybin were for the first time conclusively confirmed by HPLC, MS, and NMR to be silymarin components. Using the optimized analytical methods, six various silymarin preparations were analyzed showing substantial differences in the composition.

A Simple LC-MS Method for the Quantitation of Alkaloids in Endophyte-Infected Perennial Ryegrass.

The rapid identification and quantitation of alkaloids produced by Epichloë endophyte-infected pasture grass is important for the agricultural industry. Beneficial alkaloids, such as peramine, provide the grass with enhanced insect protection. Conversely, ergovaline and lolitrem B can negatively impact livestock. Currently, a single validated method to measure these combined alkaloids in planta does not exist. Here, a simple two-step extraction method was developed for Epichloë-infected perennial ryegrass (Lolium perenne L.). Peramine, ergovaline and lolitrem B were quantified using liquid chromatography–mass spectrometry (LC–MS). Alkaloid linearity, limit of detection (LOD), limit of quantitation (LOQ), accuracy, precision, selectivity, recovery, matrix effect and robustness were all established. The validated method was applied to eight different ryegrass-endophyte symbiota. Robustness was established by comparing quantitation results across two additional instruments; a triple quadruple mass spectrometer (QQQ MS) and by fluorescence detection (FLD). Quantitation results were similar across all three instruments, indicating good reproducibility. LOQ values ranged from 0.8 ng/mL to 6 ng/mL, approximately one hundred times lower than those established by previous work using FLD (for ergovaline and lolitrem B), and LC–MS (for peramine). This work provides the first highly sensitive quantitative LC–MS method for the accurate and reproducible quantitation of important endophyte-derived alkaloids.

Development of new and robust LC-MS method for simultaneous quantification of polyphenols from Sambucus ebulus fruits

A new and simple LC-MS method for analysis of flavonoids from Sambucus ebulus berry extracts was developed and validated. Successfully were quantitated seven polyphenols: epicatechin, epigallocatechin gallate, rutin, resveratrol, myricetin, quercetin, and kaempferol.Two detectors, working in parallel, were used: photodiode-array and single quadrupole mass-detector. The mass detection was used for identification and quantification of the analytes, while the diode-array detector was as confirmation tool. The following m/z were tracked: 457.15 (epigallocatechin gallate); 289.06 (epicatechin); 609.13 (rutin); 227.05 (resveratrol); 317.0 (myricetin); 301.02 (quercetin); 285.02 (kaempferol). For optimization the chromatographic separation three wavelengths 205 nm, 305 nm, 272 nm were monitored. The method was capable to detect in one run compounds with no UV or fluorescence chromophore and with very similar structures, such as plant polyphenols. The linearity was from 0.05 mg/L to 50 mg/L (R2 0.9962–0.9987). The recoveries for all tested analytes were between 81.6% and 104.7%.The method was applied for analysis of crude extract of Sambucus ebulus ripe fruits. Three major polyphenols – epicatechin (0.84 mg/100gFW), quercetin (0.15 mg/100gFW) and kaempferol (0.05 mg/100gFW) were identified and quantified.The proposed method could be successfully used for routine analysis of epigallocatechin gallate, epicatechin, rutin, resveratrol, myricetin, quercetin, and kaempferol in Sambucus ebulus extracts.

Semi-quantitative profiling of bile acids in serum and liver reveals the dosage-related effects of dexamethasone on bile acid metabolism in mice

Nuclear receptors, such as the pregnane X receptor (PXR) and glucocorticoid receptor (GR), play an important role in regulating the homeostasis of bile acids (BAs). In previous studies, two-week treatment of 1 mg/kg of dexamethasone (DEX) was used to activate GR in mice, whereas 4-day treatment of 75 mg/kg of DEX was chosen to activate PXR. However, little is known about the effect of DEX on circulating and hepatic BA profiles. In the present study, we reported a simple and rapid LC-MS method for semi-quantitative profiling of 39 BA species in mouse serum and liver. This method was applied to investigate the BA profiles in mice treated with either 1 mg/kg DEX for two weeks or 75 mg/kg DEX for 4 days. The gene expression, microsomal induction and liver enlargement in mice confirmed that PXR was activated by 4-day treatment of 75 mg/kg DEX, but not by two-week treatment of 1 mg/kg DEX. Two-week treatment of 1 mg/kg DEX markedly increased the circulating BAs, in particular conjugated primary BAs, suggesting a pro-cholestatic effect of DEX at low doses. In contrast, 4-day treatment of 75 mg/kg DEX increased BA hydroxylation and decreased hepatic BAs, in particular unconjugated secondary BAs, suggesting a BA-lowering and bacteria-suppressive effect of DEX at high doses. To conclude, a semi-quantitative LC-MS method was developed and applied to elucidate the dosage-related effects of DEX on serum and hepatic BA profiles in mice.

A Simple and Direct LC-MS Method for Determination of Genotoxic Impurity Hydroxylamine in Pharmaceutical compounds.

Hydroxylamine is a known genotoxic impurity compound that needs to be controlled down to ppm level in pharmaceutical processes. It is difficult to detect using conventional analytical techniques due to its physio-chemical properties like lack of chromophore, low molecular weight, absence of carbon atom and high polarity. In addition to that, analysis of the pharmaceutical samples encounters considerable obstruction from matrix components that greatly overshadow the response of hydroxylamine. This study describes a simple, sensitive and direct Liquid Chromatographic-Mass Spectrometric method (LC-MS) for detection of hydroxylamine in pharmaceutical compounds. The LC-MS method was detected up to 0.008 ppm of hydroxylamine with S/N > 3.0 and quantified up to 0.025 ppm of hydroxylamine with S/N ratio >10.0. This validated method can be applied as a generic method to detect the hydroxylamine for pharmaceutical process control and drug substance release.

Rosmarinic acid plays a protective role in the embryogenesis of zebrafish exposed to food colours through its influence on aurora kinase A level.

To evaluate the protective nature of the rosmarinic acid from Sphaeranthus amaranthoides during zebra fish embryogenesis. Rosmarinic acid was isolated from the S. amaranthoides. An accurate, sensitive and simple LC-MS analysis was performed to determine the rosmarinic acid from S. amaranthoides. In the present study, zebrafish embryos were exposed to crimson red and sunset yellow at a concentration of 0.1 and 0.5mg/l and the effect of these food colours on the levels of aurora kinase A was studied individually. Aurora kinase A levels are crucial for embryogenesis in zebrafish which is used as model in this study. The decrease of aurora kinase A levels in food colour treated embryos influences the embryogenesis, resulting in short and bent trunk leading to cell death and growth retardation. Elevated levels of aurora kinase A in rosmarinic acid treated groups can be attributed to the restoration of normal growth in zebra fish embryos with well developed brain and eyes. Further insilico docking studies were carried out and target was identified as rosmarinic acid. From the docking studies the docking poses and binding energy confirms that aurora kinase A is the target for rosmarinic acid. Rosmarinic acid was found to play a protective role in the embryogenesis of zebra fish exposed to food colours (crimson red and sunset yellow) through its influence on aurora kinase A levels.

A cross-platform metabolomics workflow for volume-restricted tissue samples: application to an animal model for polycystic kidney disease.

Metabolic profiling provides an unbiased view of the physiological status of an organism as a "function" of the metabolic composition of a measured sample. Here, we propose a simple LC-MS based workflow for metabolic profiling of volume-restricted samples, namely individual 20 μm-thick histological sections of a mouse kidney. The main idea of this workflow is to re-use the material after an RPLC-MS run, namely using the volume remaining in the vial after injection, and then introducing a phase changing step to enable HILIC-MS analysis. To test the applicability of the workflow and its ability to extract valuable biological information, we applied it to an animal model of polycystic kidney disease (PKD).

A simple LC-MS method for the determination of iohexol and iothalamate in serum, using ioversol as an internal standard

BackgroundChronic kidney disease (CKD) is diagnosed and explored through the determination of the glomerular filtration rate (GFR). Our goal was to develop a simple LC-MS method for the determination in serum of 2 popular GFR markers, contrast agents iohexol and iothalamate, for routine use and comparison studies between the two markers. A similar contrast agent, ioversol, was used as an internal standard and the method underwent a rigorous validation protocol based on β-expectation tolerance intervals. MethodsWe adapted the HPLC-UV method from Cavalier et al. to our LC-MS system. Data treatment for the validation was performed using Multiquant 3.0 (Sciex, Framingham, MA, USA) and e.noval 3.0 software (Arlenda, Liège, Belgium). ResultsAccording to the validation results our method will give accurate and reliable results for concentrations ranging from 6.8 to 250μg/ml for iohexol and 6.15μg/ml to 250μg/ml for iothalamate. In our practice these intervals are sufficient to determine both compounds in most patient samples. Samples with higher detected concentrations can always be diluted into range. ConclusionWith its internal standard and extensive validation, our method is now ready for routine and clinical research use.

Simple LC-MS Method for Differentiation of Isobaric Phosphatidylserines and Phosphatidylcholines with Deuterated Mobile Phase Additives.

Lipids from different classes sometimes can exhibit the same exact mass upon electrospray ionization; this presents an analytical challenge in lipidomics. In the negative ionization mode, for example, this can occur with phosphatidylcholines (PCs) and phosphatidylserines (PSs), making them indistinguishable in the absence of fragmentation data. PSs are found at low concentrations in biological samples, making MS/MS spectra difficult to obtain. Moreover, while PCs and PSs are distinguishable in the positive mode, PSs do not ionize as well as PCs, and their ionization is suppressed by the PCs. Here, we show that, in the negative ionization mode, substituting protiated LC-MS additives with their deuterated forms provides a way to distinguish PCs and PSs without chemical derivatization. The method described leverages the differential ionization mechanism of PCs and PSs. PCs are ionized via adduction with salts, whereas PSs ionize via hydrogen abstraction. Substituting the salts used for LC-MS with their deuterated form shifts the mass of PCs by the number of deuterium atoms in the salt, while the mass of PSs remains the same. This comparative shift enables their direct differentiation. We demonstrate that the use of deuterated formate shifts the mass of PCs and provides a direct method to distinguish PCs and PSs, even at biologically relevant low concentrations. The utility of the method was established and validated in the simultaneous analysis of PCs and PSs in lipid extracts from isolated liver mitochondria in two different rat strains. Thirteen low concentration PSs were identified that would otherwise not have been distinguishable from low concentration PCs.

A Rapid and Simple LC-MS Method Using Collagen Marker Peptides for Identification of the Animal Source of Leather.

Identification of the animal source of leather is difficult using traditional methods, including microscopic observation and PCR. In the present study, a LC-MS method was developed for detecting interspecies differences in the amino acid sequence of type I collagen, which is a major component of leather, among six animals (cattle, horse, pig, sheep, goat, and deer). After a dechroming procedure and trypsin digestion, six tryptic peptides of type I collagen were monitored by LC-MS in multiple reaction monitoring mode for the animal source identification using the patterns of the presence or absence of the marker peptides. We analyzed commercial leathers from various production areas using this method, and found some leathers in which the commercial label disagreed with the identified animal source. Our method enabled rapid and simple leather certification and could be applied to other animals whether or not their collagen sequences are available in public databases.

Comparative pharmacokinetic study of mangiferin after oral administration of pure mangiferin and US patented polyherbal formulation to rats.

The US patented polyherbal formulation for the prevention and management of type II diabetes and its vascular complications was used for the present study. The xanthone glycoside mangiferin is one of the major effector constituents in the Salacia species with potential anti-diabetic activity. The pharmacokinetic differences of mangiferin following oral administration of pure mangiferin and polyherbal formulation containing Salacia species were studied with approximately the same dose 30 mg/kg mangiferin and its distribution among the major tissue in Wistar rats. Plasma samples were collected at different time points (15, 30, 60, 120, 180, 240, 360, 480, 600, 1,440, 2,160, and 2880 min) and subsequently analyzed using a validated simple and rapid LC-MS method. Plasma concentration versus time profiles were explored by non-compartmental analysis. Mangiferin plasma exposure was significantly increased when administered from formulation compared to the standard mangiferin. Mangiferin resided significantly longer in the body (last mean residence time (MRTlast)) when given in the form of the formulation (3.65 h). Cmax values of formulation (44.16 μg/mL) administration were elevated when compared to equivalent dose of the pure mangiferin (15.23 μg/mL). Tissue distribution study of mangiferin from polyherbal formulation was also studied. In conclusion, the exposure of mangiferin is enhanced after formulation and administration and could result in superior efficacy of polyherbal formulation when compared to an equivalent dose of mangiferin. The results indicate that the reason which delays the elimination of mangiferin and enhances its bioavailability might the interactions of the some other constituents present in the polyherbal formulation. Distribution study results indicate that mangiferin was extensively bound to the various tissues like the small intestine, heart, kidney, spleen, and liver except brain tissue.