Liquid chromatography-tandem mass spectrometry validated method for the estimation of indapamide in human whole blood.

A highly precise and sensitive method for the estimation of indapamide in human whole blood using high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) is described. The method developed is validated in human whole-blood matrix, with a sensitivity of 0.5 ng/ml as lower limit of quantification. The procedure for the extraction of indapamide and glimepiride as internal standard (IS) involves haemolysis and deprotienation of whole blood using ZnSO(4) followed by liquid-liquid extraction using ethyl acetate. The sample extracts after drying were reconstituted and analysed by LC-MS/MS, equipped with turbo ion spray (TIS) source, operating in the positive ion and selective reaction monitoring (SRM) acquisition mode to quantify indapamide in human whole blood. The mean recovery for indapamide was 82.40 and 93.23% for IS. The total run time was 2.5 min to monitor both indapamide and the IS. The response of the LC-MS/MS method for indapamide was linear over the range of 0.5-80.0 ng/ml with correlation coefficient, r>or=0.9991. The coefficient of variance (% CV) at 0.5 ng/ml was 4.02% and the accuracy was well within the accepted limit of +/-20% at 0.5 ng/ml and +/-15% at all other concentrations in the linear range. This method is fully validated for the accuracy, precision and stability studies and also applied to subject-sample analysis of bioequivalence study for 1.5mg sustained-release (SR) formulations.

Rapid and sensitive method for the determination of sertraline in human plasma using liquid chromatography-tandem mass spectrometry (LC-MS/MS).

A method for the determination of sertraline, a new antidepressant drug and a selective serotonin reuptake inhibitor (SSRI), in human plasma is described. Therapeutic drug monitoring (TDM) necessitates efficient, fast and reliable analytical methods validated by external quality control. We therefore devised a simple, rapid and sensitive isocratic reversed-phase liquid chromatographic-tandem mass spectrometric method equipped with Turbo Ion spray (TIS) source, operating in the positive ion and selective reaction monitoring (SRM) acquisition mode to quantify sertraline in human plasma. A new and superior procedure of solid-phase extraction (SPE) (compared to liquid-liquid extraction) was followed to extract sertraline and imipramine as internal standard (IS) from the human plasma. Sample preparation was performed using waters hydrophilic-lipophilic balance (HLB) cartridge and this method yielded extremely clean extracts with very good recovery, 81.47 and 85.79% for sertraline and IS, respectively. Both were analyzed by combined reverse phase liquid chromatography and tandem mass spectrometry (LC-MS/MS) with positive ion TIS ionization using SRM acquisition mode. The response of the LC-MS/MS method for sertraline was linear over the dynamic range of 0.5-60.0 ng/ml with correlation coefficient r>or=0.9996. The coefficient of variance (%CV) was 8.53% at 0.5 ng/ml and the accuracy was well within the accepted limit of +/-20% at lower limit of quantification (LLOQ) and +/-15% at all the other concentrations in the linear range. This method was fully validated for the accuracy, precision and stability studies. The above findings indicate that the method is very much accurate and precise and can be successfully applied for bioequivalence studies in human subjects.

A study of serum bilirubin in neonates in relation to the maternal age.

A comparative study of neonatal serum bilirubin levels was done in neonates of different age groups of mothers. A total 122 healthy, new borns were selected for the study, born at Queen Mary's Hospital, Lucknow. Mothers were divided into two groups i.e. < 30 years and > 30 years of age. Samples of blood were collected thrice, first on day 1 from cord blood, 2nd and 3rd on days three and five of life from neonates by heel prick method, using small bore capillaries for blood collection, serum bilirubin estimation were done by the method of Malloy & Evelyn and Mean +/- SD were calculated. P-Value was observed between different age groups. In both the groups of mothers i.e. < 30 years and > 30 years serum bilirubin levels in their neonates raised to highly significant levels on day 3 (P-Value < 0.001) as compared to their cord blood serum bilirubin levels. On comparing serum bilirubin levels in neonates of both the maternal groups, it was observed that there is no significant difference between two groups on day of birth and day day 5 but statistically significant difference was observed on day 3 (P < 0.05), serum bilirubin levels were higher in neonates of younger age group mothers.

1H NMR study of the dynamics of the pH modulation of axial coordination in Aplysia limacina (Val(E7)) and sperm whale double mutant His(E7)-->Val,Thr(E10)-->Arg metmyoglobin.

The molecular and electronic structure, thermodynamics, dynamics, and mechanism of interconversion of the pH-modulated reversible equilibria of Aplysia limacina metmyoglobin, (metMb), have been investigated by 1H NMR spectroscopy. The four identified species which interconvert slowly on the NMR time scale (lifetime > 1 ms) are metMbOH (B) at alkaline pH, five coordinate metMb (N) at acidic to neutral pH, an acidic form, A, near pH approximately 4 and an extremely low pH form, D, attributed to an equilibrium unfolded species. The presence of strong distal hydrogen bonding by Arg (E10) to bound hydroxide is detected via a significant solvent isotope effect on the metMbOH (B) hyperfine shifts. Integration of the peak intensities yields pK values of 7.7 and approximately 4 for the B<-->N and N<-->A equilibria, respectively. Saturation transfer via chemical exchange is observed for B<-->N and N<-->A, where the rates for forming metMbOH (B) and the acidic form A from N are base- and acid-catalyzed, respectively, while the reverse rates are first-order. The much slower interconversion rate for N<-->B in A. limacina metMb than His(E7) containing mammalian metMb is attributed to the fact that a ligand bond is broken rather than just proton transferred and that the equilibrium involves a major rearrangement of the orientation of Arg(E10). This conclusion is supported by 1H NMR data for the sperm whale double mutant His(E7)-->Val/Thr(E10)-->Arg metMb, which exhibits a pK approximately 8.7 for the equilibrium between five-coordinate metMb (N) and metMbOH (B) with an even slower interconversion rate than in A. limacina metMb. This double mutant metMbOH (B) exhibits hydrogen bonding by Arg (E10) with coordinated hydroxide similar to that in A. limacina metMbOH. The slow but acid-catalyzed rates of conversion of A. limacina metMb (N) to the acid species A with significantly weakened bonding of the heme iron to the axial His(F8) residue is consistent with protonation of an inaccessible residue and/or a structural change accompanying the protonation equilibrium. It is concluded that metMb will coordinate water strongly only when there is a distal hydrogen bond acceptor residue, while the hydroxide ion is coordinated strongly only if there is a distal hydrogen bond donor residue.

1H-NMR study of the mechanism of assembly and equilibrium heme orientation of sperm whale myoglobin reconstituted with protohemin type-isomers.

The products of the incorporation of various protohemin type-isomers into the heme pocket of sperm whale myoglobin were investigated by 1H-NMR in the met-cyano complexes, both immediately after reconstitution as well as at equilibrium. The type-isomers studied include those involving all possible interchanges of the two substituents on a given pyrrole. The protohemin-III and -XIII isomers, with true 2-fold symmetry, yielded only homogeneous products. Protohemins-XI, -XIV both exhibited two species after reconstitution, with one disappearing with time. Protohemin-I was the only asymmetric hemin that failed to exhibit two isomers initially. The orientation of the hemin within the pocket was established by nuclear Overhauser detected dipolar connectivities among heme substituents and between heme substituents and assigned heme pocket residues. At equilibrium, the heme orientations were dominated by the asymmetric propionate rather than vinyl dispositions on the hemin, with a clear preference for placing a propionate at the 8- vs. 5-methyl position of native myoglobin. For protohemin-XI, the propionates were found in the unexpected positions of the 7-propionate and 2-vinyl groups of native myoglobin, indicating that propionates can occupy positions well within the hydrophobic interior. The alternate heme orientation for the metastable intermediates detected for protohemin-XI and -XIV involved rotational isomerism about the alpha,gamma-meso axes bisecting the vinyl positions, but these two axes are at right angles to each other in the protein matrix. The fact that protohemin-XIV, but not protohemin-I, exhibits a reversed orientation as a reconstitution intermediate provides direct evidence that vinyl contacts, as well as propionate links, modulate the relative stabilities of the initial encounter complexes between hemin and apomyoglobin. The heme cavity molecular/electronic structure was found largely unperturbed for the complexes of the various protohemin type-isomers.

Proton nuclear magnetic resonance study of the molecular and electronic structure of the heme cavity in Aplysia cyanometmyoglobin.

The 1H NMR spectrum of the low-spin, cyanide-ligated ferric complex of the myoglobin from the mollusc Aplysia limacina has been investigated. All of the resolved resonances from both the hemin and the proximal histidine have been assigned by a combination of isotope labeling, spin decoupling, analysis of differential paramagnetic relaxation, and nuclear Overhauser (NOE) experiments. The pattern of the heme contact shifts is unprecedented for low-spin ferric hemoproteins in exhibiting minimal rhombic asymmetry. This low in-plane asymmetry is correlated with the X-ray-determined orientation of the proximal histidyl imidazole plane relative to the heme and provides an important test case for the interpretation of hyperfine shifts of low-spin ferric hemoproteins. The bonding of the proximal histidine is shown to be similar to that in sperm whale myoglobin and is largely unperturbed by conformational transitions down to pH approximately 4. The two observed conformational transitions appear to be linked to the titration of the two heme propionate groups, which are suggested to exist in various orientations as a function of both pH and temperature. Heme orientational disorder in the ratio 5:1 was demonstrated by both isotope labeling and NOE experiments. The exchange rate with bulk water of the proximal histidyl labile ring proton is faster in Aplysia than in sperm whale myoglobin, consistent with a greater tendency for local unfolding of the heme pocket in the former protein. A similar increased heme pocket lability in Aplysia myoglobin has been noted in the rate of heme reorientation [Bellelli, A., Foon, R., Ascoli, F., & Brunori, M. (1987) Biochem. J. 246, 787-789].

NMR study of the molecular and electronic structure of the heme cavity of Aplysia metmyoglobin. Resonance assignments based on isotope labeling and proton nuclear Overhauser effect measurements.

The 1H NMR characteristics of the high-spin metmyoglobin from the mollusc Aplysia limacina have been investigated and compared with those of the myoglobin (Mb) from sperm whale. Aplysia metMb exhibits a normal acid----alkaline transition with pK approximately 7.8. In the acidic form, the heme methyl and meso proton resonances have been assigned by 1H NMR using samples reconstituted with selectively deuterated hemins and in the latter case by 2H NMR as well. On the basis of the methyl peak intensities and shift pattern, heme rotational disorder could be established in Aplysia Mb; approximately 20% of the protein exhibits a reversed heme orientation compared to that found in single crystals. Three meso proton resonances have been detected in the upfield region between -16 and -35 ppm, showing that the chemical shift of such protons can serve as a diagnostic probe for a pentacoordinated active site in hemoproteins, as previously shown to be the case in model compounds. The temperature dependence of the chemical shift of the meso proton signals deviates strongly from the T-1 Curie behavior, reflecting the presence of a thermally accessible Kramers doublet with significant S = 3/2 character. Nuclear Overhauser effect, NOE, measurements on Aplysia metMb have provided the assignment of individual heme alpha-propionate resonances and were used to infer spatial proximity among heme side chains. The hyperfine shift values for assigned resonances, the NOE connectivities, and the NOE magnitudes were combined to reach a qualitative picture of the rotational mobility and the orientation of the vinyl and propionate side chains of Aplysia metMb relative to sperm whale MbH2O.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of ethanol on calcium metabolism in rat erythrocyte ghosts.

A metallochromic indicator dye Arsenazo III (Az) was employed to study the effect of ethanol on calcium metabolism in rat erythrocyte ghosts. Addition of 10-150 mM ethanol to a ghost suspension induced a concentration dependent increase in calcium-Arsenazo III (Ca-Az) complex absorbance. Similar results were obtained when ghost suspensions contained (1-10 mM) theophylline. The effect of both, ethanol and theophylline was not observed if the ghosts were pre-treated with 50 microM dantrolene. Theophylline has been reported to stimulate the intracellular release of calcium in a number of cell types and the drug dantrolene is known to inhibit the intracellular release of calcium from the skeletal muscle and nerve cells. These results suggest that ethanol may release calcium from intracellular sites in these ghosts.

Determination of azo compounds with N-bromosuccinimide in acetic acid medium.

The bromination reaction of azo compounds with N-bromosuccinimide in acetic acid medium has been studied. Some reaction products have been isolated and reaction mechanisms suggested. The reaction study has been applied for the determination of 2-10 mg of azo compounds. The maximum deviation of the results from the theoretical value is generally within +/- 1%.