DEVELOPMENT AND VALIDATION OF A DRIED BLOOD SPOT LC-MS/MS ASSAY TO QUANTIFY GEMCITABINE IN HUMAN WHOLE BLOOD: A COMPARISION WITH AND WITHOUT CYTIDINE DEAMINASE INHIBITOR

Objective: The purpose of this paper is to develop and validate a LC-MS/MS method for the quantification of gemcitabine in whole human blood using dried blood spots.Methods: Gemcitabine fortified blood samples without tetrahydrouridine were spotted (50 µl) onto the DBS cards and dried for 2h at ambient room temperature. 3 mm punched spots were extracted by acetonitrile: water (90:10v/v) containing carbamazepine as internal standard (IS). Analyte and IS were separated on BDS Hypersil C18,(100 X 4.6 mm, 5 µ) column using a mixture of methanol and 2 mM ammonium acetate buffer (65:35 v/v) at a flow rate of 0.5 mL/min. Detection involved API-4000 LC-MS/MS with electrospray ionization in the positive ion mode.Results: The assay was validated over the concentration range of 5-5000 ng/ml. Intra and inters assay precision values (% CV) were less than 6.0% while the accuracy was within±15%. The mean recovery (%CV) of gemcitabine from DBS was ≥83.5% (≤4.0). Hematocrit values ranging between 0.25 and 0.62 were within acceptable limit with accuracy 93.0-103.1% of nominal values and %CV of ≤6.5 across the LQC and HQC levels. Gemcitabine was stable on DBS cards for atleast 90days at room temperature.Conclusions: A cytidine drug like gemcitabine exhibits ex vivo instability and rapidly converts to inactive metabolite in blood. All current published methods for stabilisation include tetrahydrouridine a cytidine deaminase inhibitor in the sample collection tubes. The proposed DBS method can be used as an alternative assay to conventional plasma analysis without adding enzyme inhibitor.Â

VALIDATED STABILITY-INDICATING LIQUID CHROMATOGRAPHIC METHOD FOR THE DETERMINATION OF RALOXIFENE (ANTI-OSTEOPOROTIC AGENT) IN TABLETS

Raloxifene hydrochloride is a new anti-osteoporotic agent, effective in the treatment of breast cancer. A stability-indicating high-performance liquid chromatographic method was developed and validated for the determination of Raloxifene Hydrochloride in tablet dosage forms. Reversed-phase chromatography was performed on Shimadzu Model LC-20AD Prominence  with  SPD M 20A Diode array detector using a Phenomenex Lichrosphere 100 C-18 (250 mm × 4.6 mm i.d., 5 µm particle size) column with sodium acetate: methanol (40:60, V/V) as mobile phase with a flow rate of 1 ml/min. UV detection was performed at 287 nm. Linearity was observed in the concentration range of 1.0–250 μg/mL with regression equation y = 95604 x – 26215 with correlation coefficient of 0.9999. The LOD and LOQ were found to be 0.267 μg/mL and 0.813 μg/mL respectively. The percentage relative standard deviation in precision and accuracy studies was found to be less than 2%. Raloxifene hydrochloride was subjected to stress conditions of degradation in aqueous solutions including acidic, alkaline, oxidation and photolysis. It was found that the drug is highly resistant towards all degradations as the decomposition was less than 3.5%. The developed method was validated with regard to linearity, accuracy, precision, selectivity and robustness and the method was found to be precise, accurate, linear and specific. Keywords: Raloxifene hydrochloride, Isocratic elution, RP-HPLC, Validation, Stability-indicating, LOD, LOQ

In vitro evaluation of anti oxidant activity and estimation of total flavonoids in seeds of Psoralea corylifolia plant extracts

The present study aims at in-vitro evaluation of anti oxidant activity of n-hexane and ethyl acetate extracts of seeds of Psoralea corylifolia. For evaluating this study  1, 1-diphenyl-2-picryl-hydrazyl (DPPH) method and reducing power method was used.For DPPH method Butylated Hydroxy Anisole (BHA) was used as a positive control and for  Reducing power method ascorbic acid  was used as a standard reducing agent. All the analysis was made with the use of UV-Visible Spectrophotometer. The content of total flavonoids was measured spectrophotometrically by using the aluminium chloride colorimetric assay where catechin was used as a standard. The extracts showed good DPPH radical scavenging activity and reducing power activity which was found to increase with the increasing concentration of the extracts. The total flavonoid content was found to be 73.09 mg CE/100g. Ethyl acetate extract of Psoralea corylifolia showed more prominent activity when compared to n-hexane extract which could be attributed to the presence of flavonoid content.The study could be further extended to isolate and characterize phytoconstituents from these extracts

VALIDATED STABILITY-INDICATING LIQUID CHROMATOGRAPHIC METHOD FOR THE DETERMINATION OF RALOXIFENE (ANTI-OSTEOPOROTIC AGENT) IN TABLETS

Raloxifene hydrochloride is a new anti-osteoporotic agent, effective in the treatment of breast cancer. A stability-indicating high-performance liquid chromatographic method was developed and validated for the determination of Raloxifene Hydrochloride in tablet dosage forms. Reversed-phase chromatography was performed on Shimadzu Model LC-20AD Prominence  with  SPD M 20A Diode array detector using a Phenomenex Lichrosphere 100 C-18 (250 mm × 4.6 mm i.d., 5 µm particle size) column with sodium acetate: methanol (40:60, V/V) as mobile phase with a flow rate of 1 ml/min. UV detection was performed at 287 nm. Linearity was observed in the concentration range of 1.0–250 μg/mL with regression equation y = 95604 x – 26215 with correlation coefficient of 0.9999. The LOD and LOQ were found to be 0.267 μg/mL and 0.813 μg/mL respectively. The percentage relative standard deviation in precision and accuracy studies was found to be less than 2%. Raloxifene hydrochloride was subjected to stress conditions of degradation in aqueous solutions including acidic, alkaline, oxidation and photolysis. It was found that the drug is highly resistant towards all degradations as the decomposition was less than 3.5%. The developed method was validated with regard to linearity, accuracy, precision, selectivity and robustness and the method was found to be precise, accurate, linear and specific. Keywords: Raloxifene hydrochloride, Isocratic elution, RP-HPLC, Validation, Stability-indicating, LOD, LOQ

Simultaneous quantitation of lamivudine, zidovudine and nevirapine in human plasma by liquid chromatography–tandem mass spectrometry and application to a pharmacokinetic study

A rapid and sensitive LC–MS/MS method for the simultaneous quantitation of lamivudine, zidovudine and nevirapine in human plasma using abacavir as internal standard has been developed and validated. The analytes and IS were extracted from plasma by solid phase extraction using Oasis HLB cartridges and separated on a Hypurity Advance C18 column using a mixture of acetonitrile:0.1% formic acid (76:24, v/v) at a flow rate of 0.8 mL/min. Detection involved an API-4000 LC–MS/MS with electrospray ionization in the positive ion mode and multiple-reaction monitoring for analysis. The method was validated according to FDA guidelines and shown to provide intra- and inter-day precision and accuracy within acceptable limits in a run time of only 3.5 min. The method was successfully applied to a pharmacokinetic study involving a single oral administration of a combination tablet to human male volunteers