Current Pharmaceutical Analysis - Volume 15, Issue 4, 2019

Background: Flavonoids as secondary metabolites of plants fulfill various functions in cell protection. They are of a considerable scientific interest because of their potentially medical use due to their anticancer, chemoprotective, antimicrobial, antiallergic, anti-inflammatory and antiviral activities. Objective: The study aimed to develop a new UHPLC-UV method for morin and 2 other structurally related flavonoids - naringenin and kaempferol as the structural similarity of huge numbers of flavonoids does not limit their various biological functions and activities. Methods: Separation of morin and 2 other structurally related flavonoids - naringenin and kaempferol - was achieved by using BEH C18 (1.7 μm, 2.1 x 50 mm) analytical column (Waters® Acquity UPLC) and a mobile phase composed of 0.05%v/v Formic acid in water and acetonitrile in proportion of 77:23 v/v and pumped at a flow rate of 0.4 ml/min. Column temperature was set at 25 ºC and samples were analyzed (3 μl injection volume) at a wavelength of 340 nm. Waters® Xevo G2-S QToF coupled with Waters® Acquity UPLC system with binary Solvent Manager (I-Class) via electrospray ionization (ESI) interface was used to confirm the identity of the peaks in biological samples. Results: A rapid and simple UHPLC-UV separation of morin, kaempferol and naringenin is documented including methods validation. The developed method was applied to measuring morin, kaempferol and naringenin in human plasma after a solid phase extraction. Additionally, stability of morin in tissue culture medium was verified. The extraction method and UHPLC-UV elution conditions described provide a practical means to analyze morin, kaempferol and naringenin in biological matrices. Conclusion: The developed method is fast and highly sensitive. Moreover, the flavonoids used were stable in human plasma for more than 10 days.

Background: Rivaroxaban is the first oral, selective direct FXa inhibitor with rapid onset of action and its biological toxicity may be related to the enantiomer. Objective: The aim of the current study was to develop and validate a precise, accurate, and specific direct Chiral-RP-UPLC-MS/MS method for the enantiomeric separation and detection of rivaroxaban and its enantiomer. Methods: The present study screened various conditions of chromatographic and mass spectra, including chromatographic column model, flow velocity, phase ratio, column temperature, and collision energy, parent/daughter ion pairs, etc. Try to match the chromatographic and mass spectrometric conditions. Results: Good Rs (Rs>2.5) was achieved on a Chiralpak IC column (4.6 250 mm, 5μm) using H2O:acetonitrile (10:90) as mobile phase at 25 oC column temperature. The rate of flow was set at 0.4 ml/min and enantiomers were detected by triple-quadruple tandem mass spectrometry using positive electrospray ionization (ESI) with MRM transitions of m/z 436.07>144.95. The cone voltage and collision energy were kept at 48 V and 28 eV, respectively. The limit of detection and quantification of (S)- rivaroxaban were 0.39 and 1.30 ng/ml, respectively. This method was validated and found to be selective, precise, accurate, linear and robust for the quantitative determination of chiral impurities. It is also a good application for the blood samples analysis in vitro. Conclusion: Chiral-RP-UPLC-MS/MS method has entirely detected (S)-rivaroxaban and its (R)- enantiomer in very low concentration and complex matrix directly, especially for blood samples.

Background: The therapeutic ability and application of antifungal peptide (APs) are limited by their physico-chemical and biological properties, the nano-liposomal encapsulation would improve the in vivo circulation and stability. Objective: To develop a long-circulating liposomal delivery systems encapsulated APs-CGA-N12 with PEGylated lipids and cholesterol, and investigated through in vivo pharmacokinetics. Methods: The liposomes were prepared and characterized, a rapid and simple liquid chromatographytandem mass spectrometry (LC-MS/MS) assay was developed for the determination of antifungal peptide in vivo, the pharmacokinetic characteristics of APs liposomes were evaluated in rats. Results: Liposomes had a large, unilamellar structure, particle size and Zeta potential ranged from 160 to 185 nm and -0.55 to 1.1 mV, respectively. The results indicated that the plasma concentration of peptides in reference solutions rapidly declined after intravenous administration, whereas the liposomeencapsulated ones showed slower elimination. The AUC(0-∞) was increased by 3.0-fold in liposomes in comparison with standard solution (20 mg·kg-1), the half-life (T1/2) was 1.6- and 1.5-fold higher compared to the reference groups of 20 and 40 mg·kg-1, respectively. Conclusion: Therefore, it could be concluded that liposomal encapsulation effectively improved the bioavailability and pharmacokinetic property of antifungal peptides.

Background: A chromatography tandem mass spectrometry method was first established and validated for the synchronous determination of curcumin(CUR) and paclitaxel (PTX) in this study. Objective: An LC-MS/MS Method for Determination of Paclitaxel and Curcumin. Methods: The analytes were extracted with methanol, and docetaxel was used as the internal standard (IS). The analytes and the IS were separated on a C18 (4.6 mm 50 mm, 3.5 μm) column with a mobile phase of 0.1% formic acid solution and methanol (80:20, v/v). The flow velocity of the mobile phase was 0.5 mL/min. And then, the method was applied to study the pharmacokinetic behavior of CUR and PTX in rats. Results: The calibration curves were linear within the concentration ranges of 2–1000 ng/mL for PTX and 5–500 ng/mL for CUR, the mean extraction recoveries and matrix effects of PTX, CUR, and the IS were within an acceptable range. The apparent volume of distribution of PTX was different between the group of administration of PTX and the group of co-administration with CUR and PTX. Conclusion: A sensitive and simple liquid chromatography-tandem mass spectrometry method was established and validated for the synchronous determination of PTX and CUR in rat plasma, CUR increased the apparent volume of distribution of PTX when CUR and PTX were co-administered.

Background: Ezetimibe is a lipid-lowering agent used therapeutically alone or in combination of other drugs. The properties of the solid-state of drugs are critical factors in the pharmaceutical formulation development. Several instrumental techniques can be employed in the analysis of new formulations, but the thermoanalytical techniques, provide a fast and careful evaluation of physicochemical properties of a compound. Objective: To carry out the physicochemical characterization, purity evaluation and non-isothermal kinetic studies of ezetimibe raw material. Methods: A combination of the following different analytical technics was employed: Differential Scanning Calorimetry, Thermogravimetric, Scanning Electron Microscopy, X-ray powder diffraction. Results: The results evidenced the crystalline characteristic of ezetimibe. The sample purity was 99.06 % ± 0.02 and the thermal decomposition followed a zero order kinetic, with activation energy of 96.56 kJ mol–1 and Arrhenius frequency factor of 3,442 x 109 min-1. Conclusion: The characterization of ezetimibe together with the non-isothermal kinetic degradation represents important studies for the pharmaceutical area, since it provides crucial information for the pharmacotechnical/quality control/production areas that should establish the specifications necessary to standardize the requirements of the raw material acquire to ensure the batch-to-batch reproducibility.

Background: Erythropoietin is a highly glycosylated protein hormone, used medically for the treatment of anemia associated with chronic renal disease. Due to patent expirations, we expect biosimilar erythropoietins to play an increasing role in healthcare in coming years. Objective: Here, we have developed a chromatograpy method for determination of Neu5AC and Neu5Gc in darbepoetin alfa. Methods: Sialic acids (Sias) analysis provides the contents of the two most abundant sialic acids Neu5Ac and Neu5Gc. It has often been utilized for the evaluation of sialylated glycoprotein to ensure the biopharmtherapeutic safety and efficacy. In this work, the application of the DMB derivatization of Sias from darbepoetin alfa followed by the separation of the resulting adducts based on HPLC-FLD was presented. Results: Our work demonstrated that the method is characteristics of simple operation, and high reproducibility and sensitivity, and easy to set up for compliance analysis in a routine laboratory. Conclusion: By using a water:acetonitrile gradient, high resolution of Sias was obtained within a 1 min analysis time. The detection limits of NeuAc and NeuGc were 2.9 μg/L and 1.5 μg/L, respectively. The RSDs of the intraday and interday precisions in terms of retention time and peak areas of the analytes are below 1%, and from 3.8% to 4.2% respectively. Therefore, this developed method can be widely applied for simple determination of Neu5Ac, Neu5Gc, and O-acetylated Sias in the derivatized samples. Especially, high levels of O-acetylation were detected on darbepoetin alfa N-glycans, which are critical to establishing the bioequivalence of darbepoetin alfa biosimilars.

Background: Tong-Bi-Si-Wei-Fang (TBSWF) is a candidate formula of Traditional Chinese Medicine (TCM) for treating rheumatoid bone diseases, which is composed of rhizoma corydalis alkaloids, saponins of glycyrrhiza uralensis and panax notoginseng, flavonoids of rhizoma drynariae and glycyrrhiza uralensis. Objective: Trahydropalmatine (THP), the main active ingredient of rhizoma corydalis alkaloids, was selected to study in vivo pharmacokinetics and druggability of TBSWF. Methods: The plasma concentration-time (C-T) profiles of THP and the pharmacokinetic property parameters after oral administration of THP monomer, extract of corydalis alkaloids (ECA) and TBSWF to rats, respectively were compared by a fully-validated HPLC method. Results: Compared to the THP monomer, the THP in TBSWF is absorbed faster, resides in the plasma longer and has a similar apparent volume of distribution Vz/F (10~20 L/kg). Compared to THP monomer and THP in TBSWF, the area under the concentration-time curve AUC 0-t of THP in ECA decreases two-third; Vz/F of THP in ECA (85.02 L/kg) is significantly higher than that of THP in TBSWF(p <0.05). Unlike THP monomer and THP in ECA, double peaks are observed in the C-T profile of THP after oral administration of TBSWF. THP in TBSWF exhibits slow release to a certain degree. Conclusion: The interactions among the ingredients of TBSWF promote the adsorption and prolong the residence time of THP in vivo, and provide an explanation for the advantages of TBSWF from the point of pharmacokinetics.

Background: Electrochemical oxidation mechanism and electrochemical determination of meloxicam (M), an anti-artrithtis agent, were investigated by cyclic voltammetry and square wave adsorptive stripping voltammetry, respectively. Objective: In this study, we aimed to investigate the electrochemical redox mechanism and develop a nano-sensor for sensitive, fast and selective analysis of meloxicam. Methods: In this study, the three-electrode system was used for all voltammetric measurements. Firstly, the graphene content of GR/CPE sensor was changed in the range of 1.67% to 6.68%. Then, the surface characterization of modified electrode was carried out by using Electrochemical Empedance Spectroscopy and Surface Electron Microscopy methods. Some analytical parameters, such as pH, accumulation potential and accumulation time were optimized and by using optimum parameters, calibration study was established. Results: The GR/CPE with a graphene content of 3.33 % was found to have the best voltammetric signal with a linear working range of 0.1–10 μM. The sensitivity of the quantitative voltammetric method towards M is fairly good with an LOQ of 0.0088 μmol/L and LOD of 0.0026 μmol/L. Conclusion: The optimum pH, accumulation time and accumulation potential were found to be 2.0, 150s and 0.0 V, respectively. The height of the voltammetric signal obtained with the GR/CPE electrode was stable with a 4.0 % deviation for a period of not shorter than 1 months.

Background: Midodrine (MD) is a prodrug which is converted into Desglymidodrine (DMD) after oral administration. Objective: The aim of the present study is to develop and validate a precise, accurate liquid chromatography- tandem mass spectroscopy (LC-MS/MS) method for the separation and detection of Midodrine and Desglymidodrine. Methods: The quantification of prodrug Midodrine (MD) and its active metabolite Desglymidodrine (DMD) in human plasma was performed using simple and economical protein precipitation method. Caffeine was used as an internal standard (IS). LC separation was carried out using Jones C18 column (4.6mm x 150mm, 3μm). Isocratic elution was performed using 10mM ammonium formate (pH 4.0 adjusted with formic acid): methanol 30:70, v/v as a mobile phase, at a constant of flow of 0.5 ml/min. Results: Mass spectrometric detection was carried out at positive electrospray ionization with proton adducts at m/z 255.0#131;237.1, 198.1#131;180.2 and 195.0#131;138.1 for Midodrine, Desglymidodrine and caffeine (IS) respectively, in MRM mode. The method was validated over a linear concentration range of 0.3-110 ng/ml (r2 =0.996 and r2 =0.9988) for both Midodrine and Desglymidodrine. The recovery of Midodrine and Desglymidodrine were found to be 99 ± 0.12%. The precision (intra-day and inter-day) and accuracy studies fulfilled the acceptance criteria. Conclusion: The method shows to be stable for the studied stability parameters and was successfully applied for clinical pharmacokinetic studies.

Background: Cortisol as a major glucocorticosteroid product of the adrenal cortex which has been recognized as a stress biomarker in evaluating stress related disorders for a long time. Plasma concentration of cortisol and its metabolite cortisone are usually changed in physiological and psychological tension, anxiety and depression. In order to study these changes properly, we need a sensitive, accurate and reproducible assay for plasma cortisol and cortisone determination. Objective: The aim of this study was to develop a sensitive and robust method for the determination of total cortisol and cortisone in human plasma using mass spectrometry. Methods: A fast, sensitive and selective liquid chromatography-tandem mass spectrometry (LCMS/ MS) method was developed, validated, and then the levels of cortisol and cortisone were determined. Plasma samples cleanup procedure was composed of two steps: the first was a protein precipitation with 1 % formic acid in acetonitrile, and the second was an on-line solid phase extraction (SPE). Afterwards, cortisol and cortisone were separated using a C18 ACQUITY UPLC BEHTM column with a gradient elution. The mobile phase A was 0.1 % formic acid in water, the mobile phase B was 0.1 % methanol. For the detection we used a XEVO TQ-S mass spectrometer operating in the ESI positive mode. Results: The time of analysis was 6.5 minutes and the quantification range was 5-600 ng/mL for cortisol and cortisone, with > 94% recovery for all analytes (cortisol, cortisone and internal standards). The method was validated according to the EMA guideline for bioanalytical method validation. Conclusion: A simple and sensitive LC-MS/MS method was developed and validated for measurement of cortisol and cortisone in human plasma. Our findings indicate that the proposed analytical method is suitable for routine analysis.

Background: The Rumex nepalensis Spreng (RNS) is a traditional Chinese medicine containing rich anthraquinones. However, through proper investigation we have found that there were no reports on the pharmacokinetics of RNS extract in rats. Objective: We study on the pharmacokinetic behaviors of emodin, chrysophanol and physcion after oral administration of RNS extract in rat to achieve a better understanding of further clinical application and conduct the preparation development of the herb. Methods: In the present study, a sensitive and rapid ultra-fast liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed and validated to determine the three anthraquinones such as chrysophanol, emodin and physcion in rat plasma along with danthron as the internal standard (IS). The analytes and IS were separated on an Acquity UPLC HSS T3 column (100 mm 2.1 mm, 1.8 μm) by using the mobile phase of water with 3 mM ammonium acetate and acetonitrile as gradient elution at a flow rate of 0.4 mL min -1. The detection was performed on a triple quadrupole tandem mass spectrometer equipped with electrospray ionization (ESI) by multiple reactions monitoring (MRM) of the transitions at m/z 253.1 → 225.0 for chrysophanol, 269.0 → 224.9 for emodin, 282.7→ 240.0 for physcion and m/z 239.0 → 211.0 for IS. The limit of detection and lower limit of quantification were both 2 ng mL -1 in rat plasma. Results: Good linearity of this method was obtained in the range of 2-1000 ng mL -1 , and the correlation coefficient was greater than 0.990. According to regulatory guidelines, the established method was fully validated, and the results were within acceptable limits. Conclusion: The validated method was successfully applied into a pharmacokinetic study of orally administered RNS extract in rats.

Background: 2-Cyclopentanecarbonyl-1,2,3,6,7,11b-hexahydro-pyrazino[2,1- a]isoquinolin- 4-one (P96), was found to be a novel drug candidate with one chiral center to treat schistosomiasis caused by Schistosoma japonicum. Objective: To study pharmacokinetic characteristics, a simple, rapid and sensitive liquid chromatography- tandem mass spectrometry (LC-MS/MS) method was developed and fully validated for the quantification analysis of P96 in rat plasma. Methods: Chromatographic separation was performed on a C18 column with gradient eluted mobile phase composed of acetonitrile and water at a flow rate of 0.5 mL/min. Detection was performed on a triple-quadrupole tandem mass spectrometer using positive mode electrospray ionization in the multiple reactions monitoring (MRM) mode. Results: Excellent linearity was observed in the range of 3-900 ng/mL with the lower limit of quantification of 3 ng/mL in rat plasma for P96. The intra- and inter-day precisions exhibited less than 6.6%. Mean recoveries ranged from 96.9% to 102.4%. This method was applied to investigate the enantioselective differences on the pharmacokinetics between (R,S)-P96 and its enantiomers in rats after oral administration. The enantioselective differences of (R)-P96, (S)-P96 and (R,S)-P96 were found and compared. Conclusion: The established method was found to be accurate, precise, and sensitive and can be applied to investigate the stereoselective differences on pharmacokinetics between rac-P96 and its enantiomers.

Background: Ciwujia injection, prepared from water extraction of Acanthopanax senticosus, is widely used for the treatment of cardiovascular diseases in the clinic in China. The ingredients of the preparation are still not efficiently clear and its quality control method requires further improvement. Objective: In this paper, the qualitative and quantitative methods for quality control of Ciwujia injection were originally developed by a combination of HPLC-Q-TOF-MS, HPLC-TQ-MS/MS and UPLCPDA. Methods: With HPLC-Q-TOF-MS and HPLC-TQ-MS/MS, compounds were identified firstly by comparison of their MS spectra and retention times with those of standards or recorded information in the literature. Further, a new UPLC-PDA method was originally established to simultaneously determine the multiple ingredients in the preparations. Results: For qualitative analysis, 22 compounds were identified by the presented method. For quantitative analysis, the validated method exhibited good linearity (R2 > 0.998), repeatability (RSD < 1.50%), intra- and inter-day precisions (RSD < 8.33%) and recoveries (95.93-108.58%) for the simultaneous determination of the active ingredients, including protocatechuic acid-3-glucoside, l-phenylalanine, protocatechuic acid, neochlorogenic acid, chlorogenic acid, gentiopicroside, eleutheroside B, and cryptochlorogenic acid, and was successfully utilized to analyze the eight compounds in 4 batches of Ciwujia injection. Conclusion: The presented method is simple and rapid for quality control of Ciwujia injection, and could provide a useful reference for the quality control and routine analysis of others Ciwujia preparations.

Background: Duloxetine (DL) is a selective serotonin and norepinephrine reuptake inhibitor. The drug is used in the treatment of major depression, anxiety, pain related to diabetic peripheral neuropathy and stress urinary incontinence. Objective: This study described, for the first time, the development and validation of a highly selective and sensitive microemulsion liquid chromatography-fluorescence (MELC-FL) method with low environmental pollution and without extraction steps for the simultaneous quantification of DL, and its two main metabolites; 5-hydroxy-6-methoxy duloxetine (5-HDL) and 4-hydroxy duloxetine glucuronide (4- HDLG) in plasma. Methods: The studied analytes and methyl paraben (an internal standard) were detected using excitation and emission wavelengths of 280 and 340 nm, respectively. The analysis was performed on Water Symmetry C18 analytical column (100 Å, 150 mm x 3.9 mm, 5 μm) by directly injecting the plasma after appropriate dilution with microemulsion mobile phase. Total analytical run time was 4 min. Results: The MELC-FL method was statistically validated according to the FDA guidelines for bioanalytical methods for linearity, accuracy, precision, specificity, robustness, and stability. Linear calibration plots were achieved in the ranges of 25-1200 ng/mL for DL and 50-1500 ng/mL for 5-HDL and 4- HDLG (r2 ≥ 0.997) in rat plasma. The intra- and inter- assay precisions and accuracy were acceptable. The overall recoveries of DL and its two main metabolites from rat plasma were between 97.12% and 103.12% with an RSD value between 0.34% and 4.57%. Conclusion: The present study supports the possible use of the microemulsion mobile phase in LC as a “greener ” mobile phase. The developed method offered an advantage in the form of direct analysis of biological samples after appropriate dilution with eco-friendly microemulsion mobile phase, which decreased the possibility of sample loss during analysis. The developed assay was successfully applied in a pharmacokinetic study and it established the applicability of the method for the determination of concentration-time profiles of DL and its two main metabolites in rat plasma after systemic administration.