Current Pharmaceutical Analysis - Volume 11, Issue 2, 2015

Mercaptopurine (MP) and azathioprine (AZA) are thiopurine drugs that are used in paediatrics for the treatment of inflammatory bowel disease (IBD), Therapeutic drug monitoring (TDM) of thiopurine active metabolites, 6-methylmercaptopurine (6MMP) and 6-thioguanine nucleotides (6-TGN) is a common practice for the assessment of toxicity surveillance and treatment optimization. In this paper we described the development and validation of a new High Performance Liquid Chromatography coupled to Diode Array Detection (HPLC-DAD) method for the simultaneous quantification of 6-TGN and 6-MMP in whole blood with a total runtime of 7.5 minutes over a wide range of concentrations (50 – 2000 pmol/8x10^8 RBC for 6-TG and 20 – 15000 pmol/8x10^8 RBC for 6-MMP respectively). The new method has been validated following international guidelines on bioanalytical method validation. Within- and between-run precision and accuracy results for quality control samples were always ≤ 15% of the nominal concentrations. The correlation with our reference method was excellent. The new HPLCDAD method that we describe is robust, rapid, cost effective and suitable for application to the routine TDM analyses.

Context: Liposomes appear as an excellent vehicle for parenteral and non-parenteral administration routes. Drugs, genetic vaccines or skin care products, are candidates to be included in liposomes. Irrespective of the therapeutic aim, most reported procedures for lipid vesicles formulation, make it necessary to use and then remove organic solvents. Objetive: To explore the possibility of avoiding organic solvents for liposomes preparation by sonication of component mixture. Materials and methods: Liposomes were prepared by direct sonication of lipids and water medium and by the lipid film hydration method both in the presence and absence of trehalose. Extrusion through 1.0 and 0.1 μm pore membranes was performed in both cases. Liposomes size distribution was determined by dynamic laser scattering analysis. Results: Sonication of watery lipid dispersion produces lipid vesicles in a size range of 20-120 nm while lipid film hydration method provides with larger vesicles under all experimental assayed conditions. Extrusion through 1.0 μm pore membrane failed to produce monodisperse liposomes even from the largest liposomes. Sequential extrusion though 0.1 μm, however, lead to monodisperse samples with both methods. Conclusion: The sonication of aqueous lipid dispersions is a suitable organic solvent-free alternative for preparation of small lipid vesicles.

The possible recognition interaction of R-/S-naproxen enantiomers with calf thymus DNA (ctDNA) is studied by UV absorption and fluorescence spectroscopy, KI fluorescence quenching and thermal denaturation experiments. The results indicate that ctDNA can act as host molecule to recognizing R- and S-naproxen enantiomers. As addition of ctDNA, the ρ (between 260-280 nm) and α (longer than 300 nmabsorption bands of R-/S-naproxen enantiomers show obvious hypochromic effect with bathochromic shift, indicating a typical intercalative binding mode. Importantly, the effect of ctDNA on hypochromic effect and bathochromic shift of S-naproxen is more noticeable than S-naproxen, ΔA(S) 0.57 vs ΔA(R) 0.35 at ρ band and Δλ(S) 4 nm v.s. Δλ(R) 3 nm at α band, respectively. Meanwhile, the existence of ctDNA makes the fluorescence behaviors of Rand S-naproxen enantiomers differentiable. The association constants (KA) between R-/S-naproxen and ctDNA are estimated as 1.24×104 and 1.55×104 mol·L-1, respectively, according to the Scatchard equation. Also the estimated binding sites are 0.98 and 1.0, respectively, indicating that both R- and S- naproxen could strongly and diacritically interact with ctDNA. Moreover, the Stern-Volmer fluorescence quenching constants (KSV) of R-/S-naproxen by ctDNA are measured as KS-SV 1.56×104 and KR-SV 1.41×104 mol·L-1 and their ratio, KS-SV/KR-SV, is 1.13. The fluorescence quenching by iodide anion and thermal denaturation experiments show further that the interaction mode of R-/S-naproxen and ctDNA should be an intercalative binding mode and intercalation of S-naproxen into ctDNA is stronger than R-Naproxen.

To formulate an omeprazole buccal adhesive tablet with improved bioavailability using sodium taurocholate, omeprazole buccal adhesive tablets were prepared with sodium alginate, HPMC, magnesium oxide and different quantities of sodium taurocholate. Their physicochemical properties, such as the mucoadhesive force, stability in human saliva and release, were examined. Moreover, the absolute bioavailability was assessed by comparing the buccal administration to an omeprazole solution given intravenously to hamsters. Sodium taurocholate barely influenced the mucoadhesive force of the tablets; however, it affected the drug stability in human saliva. It positively influenced the fast drug release, but did not affect the release mechanism. In particular, omeprazole buccal adhesive tablets [omeprazole/ sodium alginate/HPMC/magnesium oxide/sodium taurocholate (20/24/6/50/10 mg/tab)] showed an absolute bioavailability of about 30% in hamsters. They remained attached to the cheeks without disintegration. Moreover, they were stable in human saliva for at least 4 h. Thus, it was concluded that this omeprazole buccal adhesive tablet could be a useful possible commercial pharmaceutical product.

Generating reproducible fragmentation patterns is a problem in electrospray ionization mass spectrometry. Two generations of quadrupole time-of-flight(Q-TOF) instruments were selected to evaluate the reproducibility of tandem mass spectrometry(MS/MS) spectra:an old- and a new-generation instrument. Ninety-four MS/MS and MS/MS/MS spectra with information-rich product ions were obtained in positive- or negative-ion mode with two different-generation Q-TOF instruments, BioTOF-Q and micrOTOF- Q. Two principles were selected to optimize the collision energy. On the one hand, maximal structural information is obtained when the collision energy is increased; on the other hand, when most product ions show relative abundance of more than 10%, the lowest collision energy should be selected. The Paired-Samples T Test method was used to analysis these paired-spectra. We found that most product ions were the same and the profiles of the product ions were similar with both Q-TOF instruments. Interestingly, at the low signal to-noise ratio (S/N) of the precursor ion (ion intensity low to 180 counts), the profile of the MS/MS spectrum is very similar to that at a high S/N. Furthermore, when spectra were generated with micrOTOF-Q even before and after a four-year interval, the profiles and product ions were virtually identical. In summary, databases built with different-generation Q-TOF instruments may be universally applicable.

Aim: The purpose of this study is to explore a convenient, accurate and reliable method for quality control of Si-jun-zi Tang (SJZT) decoction. Methods: A high-performance liquid chromatography coupled with diode array and evaporative light scattering detectors (HPLC-DAD-ELSD) method and a HPLC coupled with electrospray ionization tandem mass spectrometry (HPLC-ESI-MS) method were developed for the qualitative and quantitative analysis of multiple constituents in SJZT. The chromatographic separation of multiple constituents was performed on a Boston pHlex ODS column and a C18 guard column with a gradient elution program consisting of 0.1% aqueous formic acid and acetonitrile at a flow rate of 0.7 mL/min. Results: A total of 23 constituents (7 ginsenosides, 9 licorice flavonoids, 5 licorice saponins and 2 atractylenolides) were verified based on peak retention time, ultraviolet-visible (UV) spectrum and MS data of HPLC-DAD-ELSD and HPLCESI- MS methods. The HPLC-DAD-ELSD method was successfully applied to quantify 11 major constituents (liquiritin apioside, liquiritin, Re/Rg1, isoliquiritin apioside, isoliquiritin, ononin, Rb1, Rg2, glycyrrhizic acid, and atractylenolide III) in SJZT with good linearity (R&2 > 0.9990), acceptable precision (RSD < 3.0%) and high recovery (93.4%-101.7%). Conclusion: The combined methods of HPLC-DAD-ELSD and HPLC-ESI-MS proved to be convenient, accurate and reliable methods for qualitative and quantitative analysis of SJZT.

Small differences of active pharmaceutical ingredient and excipients directly in a dosage form have been assessed on different brands of tablets of ibuprofen by a combined use of solid-state NMR and SEM offering information at both atomistic and microscopic levels of resolution. NMR spectra of pure ibuprofen and its sodium salt were measured and the chemical shifts of individual signals were used to characterize the polymorphic state of ibuprofen within tablets from five different commercial providers. Proton solid-state NMR spectra showed that ibuprofen is present in acidic form in all tablet samples and revealed different contents of bound water within tablets. Carbon NMR spectra confirmed the protonation state and furthermore ascertained the presence of the same polymorph of ibuprofen in all tablets. A single molecule of API is present in the crystallographic asymmetric unit. Solidstate NMR proved its potential in identifying small differences between tablet samples, which were attributed to different excipients whose type and quantity varied among the studied tablets. Different crystal habits have been observed for ibuprofen within tablets using scanning electron micrographs which also show variations in structure porosity of tablet samples.

The present investigation describes two simple, sensitive and validated spectrophotometric methods for the determination of Cyclophosphamide (CP) in pure and formulated forms. The methods are mainly based on the formation of charge transfer (CT) complex, with the reagents 2,3-dichloro-5,6- dicyano-1,4-benzoquinone (DDQ) (Method A) and p-Chloranilic acid (PCA) (Method B), hence a stable, 1:1 stoichiometric complex was obtained, which shows orangeyellow and pink colors, respectively. The produced chromogens optical densities were measured at 352 and 522 nm, respectively. The Beer’s law was tested in the range of 2-16 and 5-50 μg mL-1. The optical characteristics such as molar absorptivity (ɛ), and Sandell’s sensitivity values were calculated. Other analytical parameters like, Limit of Detection (LOD), Limit of Quantification (LOQ), accuracy and precisions were evaluated. The proposed methods were successfully applied for the determination of the CP in tablet formulation with higher accuracy, better recoveries and less than 2% relative standard deviation.

To accomplish a new solid dispersion providing optimized aqueous solubility and dissolution of fenofibrate without modifying the crystalline nature of the hydrophobic drug, different formulations were prepared with water, polyvinylpyrrolidone (PVP) and sodium lauryl sulphate (SLS) using the spray-drying technique. The influence of the relative quantities of PVP and SLS on the dissolution and aqueous solubility of fenofibrate in the solid dispersion was determined. The solid dispersion exhibiting highest solubility and dissolution was subjected to scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FT-IR), differential scanning calorimetry (DSC) and powder X-ray diffraction (PXRD) for physicochemical characterization. Converse to other conventional solid dispersion systems, this solid dispersion diminished the hydrophobicity of the drug by adhering hydrophilic constituents to the irregular surface of the crystalline drug during the spray drying process. In particular, the solid dispersion containing fenofibrate/PVP/SLS at a ratio of 2.5/1.5/1 (w/w/w) enormously ameliorated the aqueous solubility of fenofibrate compared to the drug powder (53.46±4.69 vs. 0.004±0.001 μg/ml). Moreover, the dissolution was approximately 75% at 30 minutes with this solid dispersion. Thus, the solid dispersion loaded with the crystalline fenofibrate might be a promising pharmaceutical product to administer poorly water-soluble drug via the oral route.

Drotaverine hydrochloride is an antispasmodic drug used to relieve cramps or spasms of the stomach, intestines and bladder. The electro reductive behaviour of drotaverine has been investigated and one irreversible well-defined cathodic peak was observed at -1.04 V vs. Ag/AgCl ((3.0 M) KCl). A validated, sensitive and reproducible cathodic adsorptive stripping voltammetric procedure for the trace determination of the bulk drug in human serum has been developed. The peak current showed a linear dependence with the drug concentration over the range 50 ng/mL to 25.6 μg/mL for Square-Wave Cathodic Adsorptive Stripping Voltammetry (SWCAdSV) and 0.2 μg/mL to 51.8 μg/mL for Differential Pulse Cathodic Adsorptive Stripping Voltammetry (DPCAdSV). Applicability to assay the drug in serum samples was illustrated and minimum detectability was found to be 49.83 ng/mL.