Current Pharmaceutical Analysis - Volume 8, Issue 3, 2012

Employment of in vitro experimentation to measure the effect of new chemical entities on human cytochrome P450 (CYP) marker activities represents a convenient approach in studying drug metabolism and pharmacokinetics. In this study, simple and accurate high performance liquid chromatograhic (HPLC) methods were developed and validated for quantitative analysis of CYP2D6-mediated dextromethorphan O-demethylation and CYP3A4-mediated testosterone 6β-hydroxylation. Both the assays showed a good linearity in the substrate concentration range of 0.05 – 20.0 μM and 0.01 – 100.0 μM with limit of detection (LOD) of 0.01 μM and 0.001 μM for CYP2D6 and CYP3A4, respectively. The intra- and inter-day precisions were from 7.21% to 12.22% and 3.09% to 14.60% for CYP2D6; and from 4.77% to 9.19% and 3.65% to 11.84% for CYP3A4. Assay accuracy for CYP2D6 ranged from 85.3% to 104.9% over dextrorphan concentrations of 0.05-5.0 μM; and that of CYP3A4 was 105.1% to 109.6% at hydroxytestosterone concentrations of 0.01-50 μM. Enzyme kinetic parameters obtained (Km and Vmax) using the two assays were within reported ranges. Thus, the assays were able to serve as activity markers in the assessment of pharmacokinetic drug interaction and metabolism mediated by CYP2D6 and CYP3A4.

High-performance liquid chromatography (HPLC) was employed in a fingerprint analysis of Radix astragali. A characteristic HPLC profile of known Radix astragali samples collected from five primary habitats in China was established by evaluating 15 samples with chemometric methods, including similarity evaluation, hierarchical cluster analysis (HCA), principal component analysis (PCA) and discriminant analysis (DA). The feasibility and advantages of employing chromatographic fingerprinting combined with chemometric methods were investigated and demonstrated in the evaluation of Radix astragali for the first time. The results showed that the chromatographic fingerprint combined with chemometric methods could efficiently distinguish Radix astragali from various areas and provide quality control in the quantitative analysis.

A dissolution test for duloxetine hydrochloride delayed release capsules was developed and validated according to current ICH and FDA guidelines. The discriminating dissolution test conditions and dissolution medium was chosen as 0.1 N HCl for acid stage and pH 6.8 buffer for buffer stage at a stirring rate of 100 rpm at 37.0°C using USP apparatus I (basket). A novel, rapid and sensitive isocratic reverse-phase HPLC method was developed for the analysis of in-vitro dissolution samples. In acidic medium, some portion of duloxetine hydrochloride degrades in to para-naphthol duloxetine and alpha-naphthol. The atomic mass numbers of degradation products formed in acidic medium were determined by LCMS/ MS. Separation of duloxetine from its major degradation impurities was achieved on Hypersil BDS C18 column (100mm x 4.6mm, 3 μm) using a 0.05 M phosphate buffer (pH 2.5)-acetonitrile (60:40 v/v) mobile phase at 40°C. The compounds were eluted isocratically at a flow rate of 1.0 mL/min and detection UV wavelength of 215 nm. In acid stage, the total % release of duloxetine was calculated as a sum of released % duloxetine and equivalent % of duloxetine converted into para-naphthol duloxetine and alpha-naphthol. During validation, standard curve was found to have a linear relationship (r > 0.998) over the analytical range of 0.02-7.5 μg/mL for acidic stage and 1.8-90 μg/mL for buffer stage. Accuracy ranged from 95 to 105 % and precision was < 2.5 % at all levels. The HPLC method was successfully applied for the screening of dissolution medium, determination of discriminatory power of dissolution test and the analysis of in-vitro dissolution samples of marketed duloxetine hydrochloride capsules.

A rapid, sensitive and selective liquid chromatography-mass spectrometry (LC-MS) method was developed and validated for the identification and determination of olanzapine (OLZ, CAS 132539-06-1) in human plasma. Following liquid- liquid extraction, OLZ and loratadine (IS, CAS 79794-75-5) were separated using a mobile phase consisting of acetonitrile: aqueous ammonium acetate solution (pH 4.0, 10 mM) = 56:44 (v/v) on an Agilent ZORBAX Eclipse XDB-CN (2.1mmx150mm I.D., 5μm) column and analyzed by electrospray ionization mass spectrometry in the selected ion monitoring (SIM) mode using the respective [M+H]+ ions, m/z 313.15 for OLZ and m/z 383.00 for loratadine. The chromatographic separation was achieved in less than 6.0 min. The assay exhibited a linear dynamic range of 0.5–50 ng/ml for OLZ in human plasma and had good accuracy and precision. Both intra- and inter-batch standard deviations were less than 15%. The validated LC–MS method was first applied to a bioequivalence study in 20 healthy male Chinese volunteers.

The objective of this study is to isolate and identify antibacterial compounds from Lithospermum erythrorhizon (L. erythrorhizon). Initially, the extracts of 44 different Traditional Chinese Medicines (TCM) were screened for their antimicrobial sensitivity by using broth micro-dilution methods on 96-microwell plate towards four different species of oral bacteria, which includes Fusobacterium nucleatum, Porphyromonas gingivalis, Streptococcus mutans and Lactobacillus acidophilus. Among the 44 TCMs, the extracts from L. erythrorhizon have proved to be most effective against these bacteria and were taken up for further study. Further, the purification of compounds from the selected L. erythrorhizon was performed by silica gel column chromatography. The compounds of L. erythrorhizon were evaluated for their efficacy by using antimicrobial sensitivity tests. Of all the extracts of L. erythrorhizon, 80% ethanol extract of it, has shown the best antibacterial effects. The elucidation of the structure of the isolated compounds was performed by NMR spectroscopy. Antibacterial effects of the compounds (Acetylshikonin, shikonin, deoxyshikonin, β-sitosterol, and β, β- dimethylacrylshikonin) obtained from the extract, showed that acetylshikonin has the best antibacterial effects. Our study concludes that acetylshikonin of L. erythrorhizon could be a potential antimicrobial agent for the treatment of oral diseases.

Ibuprofen, one of the most widely used non-steroidal anti-inflammatory drug, is an aryl acetic acid derivative, which is an active ingredient in variety of oral formulations such as tablets, gel, pellets, and syrup dosage forms used worldwide. Gastric side effects of ibuprofen are attributed to the presence of free – COOH group and inhibition of endogenous prostaglandins. In recent years, considerable research has been directed at designing prodrugs of ibuprofen with reduced gastro-intestinal toxicity. Numerous ester and amide prodrugs of ibuprofen have been reported. With this background, the present work involves the synthesis, analytical method development, in-vitro hydrolysis, bioanalytical method development, and pharmacokinetics study of an amide prodrug of Ibuprofen coded as TRB-559.

Five drugs were studied using micellar liquid chromatography (MLC) to determine micelle-water partition coefficients (Pmw) over a range of mobile phase pH values and column temperatures. In all cases the sodium dodecyl sulphate mobile phase utilised a CN reversed-phase column with UV detection, optimised for the λmax of each drug. The pH of the mobile phase was systematically varied over the range 3 to 7 pH units, incorporating values above and below the pKa‘s of the drugs studied. From this it was possible to determine MLC based values of Pmw and establish their relationships with pKa values, software predicted partition coefficients (clogP), dissociation constants (logD) and published partitioning data (logPow). This study also considered the relationship between column temperature, from 294K to 317K, and Pmw. For all five drugs it was found that Pmw decreased with increasing pH implying a systematic increased preference for the drug to remain in the aqueous phase rather than partition into the micellar phase. In addition, the partition coefficient displayed a linear relationship with log D over the pH range for each drug with a ‘break-point’ observed at the pKa for each drug. With respect to increasing temperature, the results were non-linear indicating that there is no general relationship for these drugs with temperature. Overall, it was found that MLC is suited to the measurement of partition coefficients for pharmaceutical compounds yet it should be noted that both pH and temperature play a significant role in the values obtained.

Two novel, simple and rapid stability-indicating spectrofluorimetric methods have been developed for the determination of sitagliptin in tablets and spiked serum samples. In the first method, sitagliptin`s natural fluorescence was measured at 353 nm after excitation at 259 nm. On the basis of the reaction between sitagliptin and fluorescamine the second method has been developed in borate buffer solution of pH 9.0 and the fluorescence intensities of the derivatives were measured at 475 nm emission and 390 nm excitation wavelengths. Thecalibration curves were constructed in concentration range of 0.5–10.0 μg mL-1 and 0.2-1.4 μg mL-1 for the first and second method, respectively. The developed methods were validated with respect to linearity, precision, sensitivity, accuracy and selectivity. The degradation behaviour of the drug was investigated using the first method. The drug solution was subjected to neutral, acid and alkali hydrolysis, oxidation, thermal stress and exposured to the sunlight. The first method is proved to be selective and useful for the investigation of the stability of sitagliptin. The application of spiked serum samples was also analysed by using the second method. The developed methods were successfully applied for the determination of sitagliptin in tablets and spiked serum samples.

To assess the interaction of mefloquine (MQ) with biomembranes and serum albumin, the partitioning of MQ into phosphatidylcholine-phosphatidylserine small unilamellar vesicles (PC-PS SUV) and its binding with bovine serum albumin (BSA) were examined using various spectroscopic methods. The partition coefficients (Kps) of MQ, determined by the second-derivative spectrophotometric method, increased in accordance with the PS content in PC-PS SUV: the Kp values for PC-PS SUV with PS contents of 10 mol% were about 2.5 times that for PC SUV. The zeta potential values of PC-PS SUV fell from -10 mV at a PS content of 0 mol% to -42 mV at PS contents of 10 mol%, and were found to depend to some extent on the Kp values. Meanwhile, the binding constant (logK) and the number of binding sites (n) for the binding of MQ to BSA were determined using a fluorescence quenching method. These values were significantly reduced by the presence of Cl– ions, from 5.55 to 4.66 for logK and from 1.30 to 1.11 for n. To examine the binding site of MQ on BSA, a 19F nuclear magnetic resonance (NMR) spectroscopic study was performed using typical competing ligands bound to BSA. The results revealed that whereas most of the MQ binds nonspecifically to BSA, MQ also partially binds to Site II. Finally, these results showed that the major interactions between MQ and PC-PS SUV or BSA were essentially different; the partitioning of MQ into PC-PS SUV was electrostatic and the binding of MQ to BSA was hydrophobic.

A novel liquid chromatographic–electrospray ionization mass spectrometric (LC–ESI-MS) method has been developed for the determination of Armodafinil in human plasma using carbamazepine as internal standard. The sample was prepared by employing liquid–liquid extraction method from human plasma using ethyl acetate as a solvent. The chromatographic separation was achieved within 3.0 min by using 0.2% formic acid: methanol (15:85) as mobile phase on hypurity advance C-18 column (5μ; 100 x 4.6 mm) at a flow rate of 1.0 ml/min. Ion signals m/z “274.1/167.3, 237.0/192.0” for armodafinil and internal standard respectively were measured in the positive ion mode. A detailed validation of the method was performed as per US-FDA guidelines (ICH Q2B). The results of all validation parameters were found to be within the acceptance limits. The drug concentration range from 50-10000 ng/mL was shown to be linear (r2 = 0.9989). Accuracy of the method was found to be >947percnt;, and lower limit of quantification was found at 50 ng/mL. The extraction recoveries were found to be 70.6±0.96% and 67.7±1.32% for ARM and IS, respectively. The recoveries of the stability of sample at different conditions were found to be more than 95%. From the results, it is suggested that the proposed method is simple, reproducible, accurate and precise. So, this method can be applied for the future investigative studies of ARM in human plasma.