Seasonal influence on expression of heat shock proteins (HSP70 and HSP90) vis-à-vis functional competence of Gir bull semen.

The success of assisted reproduction relies on functional competence of frozen-thawed semen. Heat stress affects protein folding leading to aggregation of mis-folded proteins. Hence, a total of 384 (32 ejaculates/bull/season) ejaculates from six matured Gir bulls were used to evaluate physico-morphological parameters, the expression of HSPs (70 and 90) and fertility of frozen-thawed semen. The mean percent individual motility, viability and membrane integrity were significantly (p < 0.01) higher in winter compared to summer. Out of 1200 Gir cows inseminated, 626 confirmed pregnant and the mean conception rate of winter (55.04 ± 0.35) was significantly (p < 0.001) higher than summer (49.33 ± 0.32). A significant (p < 0.01) difference in concentration of HSP70 (ng/mg of protein) but not HSP90was observed between the two seasons. The HSP70 expression in pre-freeze semen of Gir bulls had significant positive correlation with motility (p < 0.01, r = 0.463), viability (p < 0.01, r = 0.565), acrosome integrity (p < 0.05, r = 0.330) and conception rate (p < 0.01, r = 0.431). In conclusion, the season influences physico-morphological parameters and expression of HSP70 but not HSP90 in Gir bull semen. The HSP70 expression is positively correlated with motility, viability, acrosome integrity and fertility of semen. The semen expression of HSP70 may be utilized as biomarker for thermo-tolerance, semen quality and fertilizing capacity of Gir bull semen.

Determination of ergocalciferol in human plasma after Diels-Alder derivatization by LC-MS/MS and its application to a bioequivalence study.

An accurate, sensitive and selective method is developed for determination of ergocalciferol (vitamin D2) in human plasma using LC-MS/MS. After liquid-liquid extraction with n-hexane, ergocalciferol was derivatized by reacting with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD), a strong dienophile based on Diels-Alder reaction. Ergocalciferol and its deuterated internal standard, ergocalciferol-d6, were analyzed on X Select CSH C18 (100 mm×4.6 mm, 2.5 µm) column using acetonitrile and 0.1% (v/v) formic acid in water containing 0.14% methylamine within 6.0 min under gradient elution mode. Tandem mass spectrometry in positive ionization mode was used to quantify ergocalciferol by multiple reaction monitoring (MRM). Entire data processing was done using Watson LIMS™ software which provided excellent data integrity and high throughput with improved operational efficiency. The major advantage of this method includes higher sensitivity (0.10 ng/mL), superior extraction efficiency (≥83%) and small sample volume (100 µL) for processing. The method was linear in the concentration range of 0.10-100 ng/mL for ergocalciferol. The intra-batch and inter-batch accuracy and precision (% CV) values varied from 97.3% to 109.0% and 1.01% to 5.16%, respectively. The method was successfully applied to support a bioequivalence study of 1.25 mg ergocalciferol capsules in 12 healthy subjects.

Determination of ergocalciferol in human plasma after Diels-Alder derivatization by LC–MS/MS and its application to a bioequivalence study / 药物分析学报

An accurate, sensitive and selective method is developed for determination of ergocalciferol (vitamin D2) in human plasma using LC–MS/MS.After liquid-liquid extraction with n-hexane,ergocalciferol was derivatized by reacting with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD), a strong dienophile based on Diels-Alder reaction. Ergocalciferol and its deuterated internal standard, ergocalciferol-d6, were analyzed on X Select CSH C18 (100 mm×4.6 mm, 2.5μm) column using acetonitrile and 0.1% (v/v) formic acid in water containing 0.14% methylamine within 6.0 min under gradient elution mode. Tandem mass spectrometry in positive ionization mode was used to quantify ergocalciferol by multiple reaction monitoring(MRM).Entire data processing was done using Watson LIMS? software which provided excellent data integrity and high throughput with improved operational efficiency.The major advantage of this method includes higher sensitivity(0.10 ng/mL), superior extraction efficiency(≥83%)and small sample volume(100μL)for processing.The method was linear in the concentration range of 0.10–100 ng/mL for ergocalciferol.The intra-batch and inter-batch accuracy and precision (% CV) values varied from 97.3% to 109.0% and 1.01% to 5.16%, respectively. The method was successfully applied to support a bioequivalence study of 1.25 mg ergocalciferol capsules in 12 healthy subjects.

Selective and rapid determination of raltegravir in human plasma by liquid chromatography-tandem mass spectrometry in the negative ionization mode.

A selective and rapid high-performance liquid chromatography-tandem mass spectrometry method was developed and validated for the quantification of raltegravir using raltegravir-d3 as an internal standard (IS). The analyte and IS were extracted with methylene chloride and n-hexane solvent mixture from 100 µL human plasma. The chromatographic separation was achieved on a Chromolith RP-18e endcapped C18 (100 mm×4.6 mm) column in a run time of 2.0 min. Quantitation was performed in the negative ionization mode using the transitions of m/z 443.1→316.1 for raltegravir and m/z 446.1→319.0 for IS. The linearity of the method was established in the concentration range of 2.0-6000 ng/mL. The mean extraction recovery for raltegravir and IS was 92.6% and 91.8%, respectively, and the IS-normalized matrix factors for raltegravir ranged from 0.992 to 0.999. The application of this method was demonstrated by a bioequivalence study on 18 healthy subjects.

Selective and rapid determination of raltegravir in human plasma by liquid chromatography-tandem mass spectrometry in the negative ionization mode / 药物分析学报

A selective and rapid high-performance liquid chromatography–tandem mass spectrometry method was developed and validated for the quantification of raltegravir using raltegravir-d3 as an internal standard (IS). The analyte and IS were extracted with methylene chloride and n-hexane solvent mixture from 100 mL human plasma. The chromatographic separation was achieved on a Chromolith RP-18e endcapped C18 (100 mm ? 4.6 mm) column in a run time of 2.0 min. Quantitation was performed in the negative ionization mode using the transitions of m/z 443.1-316.1 for raltegravir and m/z 446.1-319.0 for IS. The linearity of the method was established in the concentration range of 2.0–6000 ng/mL. The mean extraction recovery for raltegravir and IS was 92.6% and 91.8%, respectively, and the IS-normalized matrix factors for raltegravir ranged from 0.992 to 0.999. The application of this method was demonstrated by a bioequivalence study on 18 healthy subjects.

Determination of (S)-(+)- and (R)-(-)-ibuprofen enantiomers in human plasma after chiral precolumn derivatization by reversed-phase LC-ESI-MS/MS.

BACKGROUND: A selective, sensitive and high-throughput LC-ESI-MS/MS method has been developed and validated for the chromatographic separation and quantitation of (S)-(+)-ibuprofen and (R)-(-)-ibuprofen after derivatization with (S)-(-)-1-(1-napthyl)ethylamine using 1-hydroxybenzotriazole as the activator of the carboxylic acid group and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide as the coupling reagent in human plasma. RESULTS: Both the analytes were chromatographically separated with a resolution factor of 1.27 on a Kinetex PFP (50 × 4.6 mm, 2.6 µm) analytical column. The method was validated over the concentration range of 0.10-32.0 µg/ml for both the enantiomers. The magnitude of matrix effect was assessed by post-column analyte infusion and also by precision (%CV) values for the calculated slopes of calibration curves. The mean extraction recovery was >91% for both the enantiomers. CONCLUSION: The method was successfully applied to a bioequivalence study in 34 healthy human subjects. The assay reproducibility was confirmed by reanalysis of 130 subject samples.

Enhanced Fresnel zone plate coded microscopy of large-size objects.

A scheme for microscopy of relatively large-size objects by using Fresnel zone plate (FZP) coded imaging (FZ-PCI) is digitally demonstrated. The limit on the source size in zone-plate-based microscopy comes from interference of out-of-focus multidiffraction orders of the FZP with the focused-order image. From the study of the angular spectrum of the coded image, it is shown that noise contribution from higher orders to a lower-order image can be digitally suppressed by selective propagation of spatial frequencies. Similarly, noise from aliasing and noise from lower orders to a higher-order image can be reduced by spatially limiting the coded image. To my knowledge for the first time, the results of digitally performed FZPCI-based microscopy of an object that is three times larger than the first zone of the FZP with a resolution better than 2 microm are presented and discussed.