KULDEEP SHARMA

... Thermophysical and structural studies on some glass-ceramics and role of nano size crystallites GP Kothiyal*, Arvind A, Rakesh Kumar, Anupam Dixit, Kuldeep Sharma and Madhumita Goswami ... Res. 2 117-41. [3] Singh RK, Kothiyal GP and Srinivasan A 2008 J. Non-Cryst. ...

Simmondsia chinensis (Link) Schneider, a multipurpose dioecious shrub of arid zones, has emerged as a cash crop. It is being cultivated for its seeds which store liquid wax whose properties are similar to spermaceti (Sperm whale oil), a substitute for petro products and precious high-priced lubricants. Jojoba is a slow-growing desert shrub having a male biased (5:1; male:female ratio) population. Since there is no method available to determine the sex at the seedling stage, current investigations have been carried out to generate a sex-specific random amplified polymorphic DNA (RAPD) marker in jojoba which is based on the PCR amplification of random locations in the genome of plant. Of the 72 primers tested, only one random decamer primer, OPG-5, produced a unique ∼1,400 base pairs fragment in male DNA. To validate this observation, this primer was re-tested with the individuals of male and female samples of four cultivars. The unique ∼1,400 bp fragment was present in male individuals of all the four cultivars and completely absent in respective female individuals tested. To the best of our knowledge, this is the first report to ascertain the sex of jojoba plants at an early stage of development of the taxon.

Anemias are the blood disorders characterized by reduction in the number of circulating red blood cells, the amount of hemoglobin, or the volume of packed red cells in blood. Chromosomal aberrations have often been reported from the bone marrow as well as cultured lymphocytes of the anemic patients. The aims of the study were to find out the commonest type of anemia occurring in the population of Jammu, India and to find out the chromosomal changes involved in the disorder. Present study has been carried out on the bone marrow samples from 53 clinically diagnosed anemic patients. Cytogenetic study was carried out on slides prepared from these samples. Noncytogenetic factors like age, sex, religion, blood groups, family history of anemia, socioeconomic status, etc. have also been included in the study. Megaloblastic anemia was found to be the commonest type of anemia. Centromere stretching, chromatid breaks, gaps, and elongation of chromosomes were recorded in patients with megaloblastic anemia and combined deficiency anemia. However, structural changes and numerical changes were totally absent. The commonest anemia among the people of Jammu region is megaloblastic anemia and its prevalence is increasing every year. Also, megaloblastic anemia is always associated with reversible cytogenetic changes.

A novel High Performance Liquid Chromatography — electrospray mass spectrometric method has been developed for the estimation of Ursodiol (Ursodeoxycholic acid)-a bile acid, in human plasma using Ornidazole as internal standard. The methodology involved solid phase extraction of the analyte from human plasma matrix. The chromatographic separation was achieved within seven minutes by an isocratic mobile phase containing 1.0 mM ammonium acetate and Acetonitrile (65: 35, v/v), flowing through XTerra MS C18, 100 × 2.1, 3.5 μm analytical column, at a flow rate of 0.2 ml/min. Ion signals were measured in negative mode for Ursodiol and internal standard at m/z 391.3 and 278.1, respectively. A detailed validation of the method was performed as per USFDA guidelines and the standard curves were found to be linear in the range 50.0 ng/ml to 3000.0 ng/ml with the mean correlation coefficient more than 0.99. The absolute recovery was more than 54.90 % for Ursodiol and 76.51 % for internal standard. Ursodiol was stable for sixty-nine days at −70 °C and for eight hours at ambient temperature. After extraction from plasma, the reconstituted samples of Ursodiol were stable in autosampler at 10 °C for forty-eight hours. Upon subjecting to three freeze thaw cycles, there was no change in the recovery of the analyte. The integrity of the plasma samples remained unaffected even upon four-fold dilution with drug free human plasma. The method was simple, specific, sensitive, precise, accurate and suitable for bioequivalence and pharmacokinetic studies. It was successfully applied to the pilot bioequivalence study of Ursodiol in male human subjects.

In vitro clonal propagation of 18–20-yr-old Holarrhena antidysenterica tress has been achieved by employing nodal explants. The tree explants showed marked seasonal variation in their morphogenic response under in vitro conditions. Maximum response was obtained from the beginning of May to the end of July, followed by a gradual decline, finally dropping to zero from October to February. The explants induced multiple shoots only on cytokinin-containing medium. Several cytokinins [N6-benzyladenine (BA), N6-(2-isopentenyl) adenine (2ip), 6-furfuryl aminopurine (Kn), and adenine sulfate (Ads)] were assayed. The best response was achieved with 15 μM BA in which 62.5% of cultures produced 2.75±0.2 shoots per explant with 3.56±0.2 cm average length. Amongsth the three heavy metals assayed, silver nitrate (AgNO3) significantly improved the response. This compound enhanced both the percentage of responding cultures (86.6%) and the average shoot number (4.73±0.2) at a concentration of 20mgl−1. Further improvement in the morphogenic response occurred when explants from in vitro shoots were employed instead of mature trees. In this case, the percentage of morphogenic cultures was increased to 100% at the third subculture with an average of 11.45±0.3 shoots per explant. Regenerated shoots were rooted in half-strength Murashige and Skoog medium with 10 μM indole-3-acetic acid. The plantlets were successfully acclimatized in soil.

A new method was developed for determination of itopride in human serum by reversed phase high-performance liquid chromatography (HPLC) with fluorescence detection (excitation at 291 nm and emission at 342 nm). The method employed one-step extraction of itopride from serum matrix with a mixture of tert-butyl methyl ether and dichloromethane (70:30, v/v) using etoricoxib as an internal standard. Chromatographic separation was obtained within 12.0 min using a reverse phase YMC-Pack AM ODS column (250 mm x 4.6 mm, 5 microm) and an isocratic mobile phase constituting of a mixture of 0.05% tri-fluoro acetic acid in water and acetonitrile (75:25, v/v) flowing at a flow rate of 1.0 ml/min. The method was linear in the range of 14.0 ng/ml to 1000.0 ng/ml. The lower limit of quantitation (LLOQ) was 14.0 ng/ml. Average recovery of itopride and the internal standard from the biological matrix was more than 66.04 and 64.57%, respectively. The inter-day accuracy of the drug containing serum samples was more than 97.81% with a precision of 2.31-3.68%. The intra-day accuracy was 96.91% or more with a precision of 5.17-9.50%. Serum samples containing itopride were stable for 180.0 days at -70+/-5 degrees C and for 24.0 h at ambient temperature (25+/-5 degrees C). The method was successfully applied to the bioequivalence study of itopride in healthy, male human subjects.

The nodal explants of in vitro shoots of Holarrhena antidysenterica L. were cultured on Murashige and Skoog's (MS) medium augmented with 15 μM N6-benzyladenine (BA) alone (control) or supplemented with different concentrations (1, 5, 10 and 20 mg dm−3) of CdCl2, CuSO4, Pb(NO3)2 and ZnSO4. The maximum morphogenic response in terms of average shoot number (4.95 ± 0.17) was seen in control. ZnSO4 proved to be less inhibitory in comparison to CuSO4, Pb(NO3)2 and CdCl2. None of the explants cultured on CdCl2 containing medium induced multiple shoots. Maximum protein content [3.80 ± 0.04 mg g−1(f.m.)] was observed in control and slightly less [3.50 ± 0.02 mg g−1(f.m.)] in tissues exposed to 1 mg dm−3 of CuSO4 and minimum [1.00 ± 0.02 mg g−1(f.m.)] in Zn treated (20 mg dm−3) explants. SDS-PAGE analysis of the treated tissues revealed that two new polypeptides (29 and 20 kDa) in response to Cu and Zn treatment, respectively, have been synthesized.

A new HPLC method was developed for the estimation of carboxylic acid metabolite of clopidogrel bisulfate in rat plasma using atorvastatin as internal standard. Plasma samples were extracted with a mixture of ethyl acetate and di-chloro methane (80:20, v/v) followed by subsequent reconstitution in a mixture of water:methanol:acetonitrile (40:40:20, v/v). The chromatographic separation was achieved with gradient elution on Kromasil ODS, 250 mm x 4.6 mm i.d., 5 microm analytical column maintained at 30 degrees C. Carboxylic acid metabolite of clopidogrel as well as the internal standard were detected at a wavelength of 220 nm. The method was validated as per USFDA guidelines. Calibration curves were linear in the concentration range of 125.0-32,000 ng/ml and the correlation coefficient was better than 0.999. The extraction efficiency for the carboxylic acid metabolite of clopidogrel was more than 85.76%. The intra-day accuracy ranged from 98.9% to 101.5% with a precision of 1.30% to 6.06%. Similarly, the inter-day accuracy was between 96.2% and 101.1% with a precision of 3.47% to 4.30%. The drug containing plasma samples were stable at -70 degrees C for 48 days and at ambient temperature for 24h. In the auto-sampler maintained at 15 degrees C, the processed and reconstituted samples were stable for 35 h. The drug containing frozen plasma samples were stable enough to with stand three freeze thaw cycles. The method was successfully applied to the pharmacokinetic study of the two different polymorphs of clopidogrel bisulfate in Wistar rat.

Simmondsia chinensis (Link) Schneider, a multipurpose and monogeneric dioecious shrub from arid zones, has emerged as a cash crop all over the globe. Its seed propagation poses severe problems due to its male-biased population: the male:female ratio is 5:1. Investigations have been carried out to generate a sex-specific Inter-simple sequence repeat (ISSR) marker for the early detection of male and female plants. Of the 42 primers analysed with a bulk sample of pooled male DNA and a bulk sample of pooled female DNA, only one primer, UBC-807, produced a unique ~1,200 base-pair fragment in the male DNA. To validate this observation, this primer was re-tested with individual male and female samples from eight cultivars. A similar unique ~1,200 bp fragment was present in the male individuals of all eight cultivars and completely absent in the female individuals tested. This is the first report of the use of ISSR markers to ascertain sex in physiologically mature S. chinensis plants.

A LC-MS/MS method has been developed for the estimation of rosuvastatin in human plasma using atorvastatin as internal standard. Rosuvastatin is a lipid-lowering drug prescribed for the treatment of hyper-cholestrolemia and dyslipidimia. Solid phase extraction (SPE) was used for the purification and pre-concentration of analyte from human plasma matrix. The chromatographic separation was achieved within 6.0 min by an isocratic mobile phase containing 0.2% formic acid in water and acetonitrile (40: 60, v/v), flowing through YMC J' Sphere ODS H-80, 150 x 4.6 mm, 4.0 µm analytical column, at a flow rate of 1.0 mL min-1 with split of 200  µL to mass spectrometer and 800  µL to waste. Multiple reaction monitoring (MRM) transitions were measured in the positive mode at m/z 482 and 258 for rosuvastatin and m/z 559 and 440 for internal standard respectively. A detailed validation of the method was performed as per USFDA guidelines and the standard curves were found to be linear in the range 1.0 ng mL-1 to 50.0 ng mL-1 with the mean correlation coefficient more than 0.99. The absolute recovery was more than 50.14% for rosuvastatin and 54.65% for internal standard. In human plasma, rosuvastatin was stable for 138 days at –70 ± 5 °C and for 24 hours at ambient temperature. After extraction from plasma, the reconstituted samples of rosuvastatin were stable in auto sampler at 10 °C for 8 hours. Upon subjecting to three freeze thaw cycles, there was no change in the recovery of the analyte. The method was simple, specific, sensitive, precise, accurate and suitable for bioequivalence and pharmacokinetic studies. It was successfully applied to the pilot bioequivalence study of rosuvastatin 20 mg tablets of M/s Zydus Cadila health care Ltd. India versus 20 mg Crestor tablet of M/s Astra Zeneca, USA; in male human subjects.