Current Pharmaceutical Analysis - Volume 16, Issue 5, 2020

Darunavir and Ritonavir are amongst the most useful antiretroviral drugs worldwide for treating AIDS (acquired immune deficiency syndrome). This review discussed and summarized the various analytical techniques used in the qualitative and quantitative analysis of darunavir and ritonavir, protease inhibitors (PIs), which have gained importance as antiretroviral drugs. The importance and use of chromatographic techniques for the estimation of darunavir and ritonavir are also discussed herein. This review highlights the various advanced analytical techniques such as spectroscopic, chromatographic, electrophoresis, X-ray powder diffraction and their corresponding methods.

Chiral separation plays a very important role in the modern pharmaceutical analysis and will continue in upcoming years. Separation and identification of chiral impurities are indispensable. According to ICH guidelines, only the active enantiomer of the drug has to be marketed, so there is a focus on separation of the inactive enantiomer which acts as a chiral impurity. The impurities present in the enantiomers also pose various toxic adverse effects on bioavailability and efficacy, hence the need to separate these impurities will forever be trending. This review primarily focuses on the separation techniques like Capillary Electrophoresis (CE), High-Performance Liquid Chromatography (HPLC), Gas Chromatography (GC), and Supercritical Fluid Chromatography (SFC) followed by the year-wise trend in the separation of selected chiral impurities. In the coming years, researchers should work on using ultra-fast, selective, and sensitive methods for the effective separation of chiral impurities.

Background: Venlafaxine (VEN) is a bicyclic phenylethylamine derivative and possesses a marked structural difference from other antidepressant drugs present in the market. It works by eliciting the neurotransmitter action in CNS. It occurs in two enantiomeric forms i.e. R and S VEN. After the first pass metabolism, it gets metabolized into more active form O-desmethylvenlafaxine (ODV) which also exist in the enantiomeric forms. So it is important to develop a suitable analytical and bioanalytical method for the determination of VEN and its metabolite to quantify them accurately. Methods and Results: The current review summarizes methods to determine chiral and achiral forms of VEN and ODV. According to the literature, it is clear that most widely used method for the determination of VEN and ODV is liquid chromatography-mass spectroscopy, other methods used for routine analysis include UV spectroscopy, reverse phase high-performance liquid chromatography with PDA detector. For the determination of enantiomeric forms of VEN and ODV, different chiral columns have been utilized. Capillary electrophoresis with charged cyclodextrins is also used to determine the enantiomeric forms. Conclusion: Various analytical methods for determining VEN and its metabolite in different matrices have been discussed thoroughly in the present review.

Background and Objective: The present study describes the UPLC-MS/MS method validation for the analysis of ornidazole in solid dispersion. Methods: The proposed UPLC-MS/MS method utilizes BEH Shield RP18 column (2.1 mm 100 mm, 1.7 μm) with a gradient programmed mobile phase composed of water and acetonitrile at a flow rate of 0.4 mL/min which varies with time program. Ornidazole was detected by UPLC-MS/MS with three proton adducts at m/z 82.04, 128.05 as daughter ions and 220.03 as a parent ion in Multiple Reaction Monitoring (MRM) operated in positive mode. Results: Adducts at m/z 128.05 was found to be the most stable and showed higher intensity was selected for quantification of ornidaozle in solid dispersion. In the method, validation linearity was determined at concentration range of 10-100ng/mL and a correlation coefficient was found (r2) ≥0.9994. The limit of detection and limit of quantification were found to be 1.5 and 4 ng/mL, respectively. Inter and Intra-day precision was found within 0.33 and 0.11% and accuracy within 100.08% and 100.04%. Conclusion: A sensitive and selective UPLC-MS/MS method had been validated for the analysis of ornidazole in solid dispersion. The proposed method of analysis of ornidazole in solid dispersion can be used in quality control laboratories.

Background: Convolvulus species are extensively used in traditional medicines for the treatment of peptic ulcer diseases primarily associated with the presence of many bioactive compounds, such as coumarins. Methods: A validation and improvement of a rapid, sensitive and simple technique for bioactive compounds analysis of scopolin and scopoletin in Convolvulus pilosellifolius (CP) and Convolvulus austroaegyptiacus (CA) plant extracts using UHPLC-MS/MS were performed. Both plants extracts were subjected to high-resolution DART-ToF-MS technique for fast profiling of their constituents. Results: DART-ToF-MS spectra proved the presence of experimental mass at 193.05137 and 355.10371 m/z in the extract of CA plant and at 193.05084 and 355.10365 m/z in CP plant extract for scopolin and scopoletin compounds, respectively. The total chromatographic analysis time was less than 10 min with excellent resolution and negligible matrix effect. The validated parameter showed good linearity (R2 ≥0.998) over a wide concentration range of 0.05-10.0 μg mL-1 for both scopolin and scopoletin, detection limits of scopolin and scopoletin were 0.03 and 0.01 μg mL-1, respectively. The method also showed good intraday and interday precisions (RSD<4.33%). The recovery percentage values were between 97.04 and 99.97% at three added concentration limits. The highest content of scopolin (3.485 mg g-1) and scopoletin (0.795 mg g-1) was detected in CA ethanolic extract. The studied plants were finally compared with all previous reports in terms of scopolin and scopoletin content. Conclusion: The results indicate that the suggested method can be used for regular analysis of active compounds in medicinal plant extracts.

Background: Periplogenin, an active ingredient in Cortex Periplocae, is widely noted for its multiple biological activities; however, the metabolism of this compound has been scarcely investigated. The present report proposed the in vitro metabolic profiling and reaction pathways of periplogenin in rat liver microsomes. Method and Results: The metabolites of periplogenin in rat liver microsomes were analyzed. Two main metabolites, namely 14-hydroxy-3-oxo-14β-carda-4, 20 (22)-dienolide and 5, 14-dihydroxy-3-oxo-5β, 14β-card-20(22)-enolide were identified by HPLC-MSn, 1H-NMR and 13C-NMR. HPLC method was established for the simultaneous determination of periplogenin and its related metabolites (M0, M1 and M2), which was performed on Waters ODS column with a methanol-water solution (53:47, v/v) as mobile phase and descurainoside as an internal standard at 220 nm. The linearity ranges of M0, M1 and M2 were 0.64-820.51, 0.68-864.86 and 0.64-824.74 μM respectively with the regression coefficient (R2) above 0.9995. The limits of quantitation for these metabolites (M0, M1 and M2) were 0.18, 0.22 and 0.15 μM respectively. The developed method was also accurate (with relative errors of -3.6% to 3.2%) and precise (with relative standard deviations below 7.9%). The recoveries of the three analytes were above 85.7% with stability in the range of -2.4% to 3.6%. The enzyme-kinetic parameters of periplogenin including Vmax (6.08 ± 0.19 nmol/mg protein/min), Km (288.62 ± 14.54 μM) and Clint (21 ± 1.0 μL/min/mg protein) were calculated using nonlinear regression analysis. Conclusion: These findings significantly highlighted the metabolic pathways of periplogenin and also provided some reference data for future pharmacokinetic and pharmacodynamic studies.

Background: The high resolving and accuracy power of the HRMS instrument enabled us to identify the product ions and to propose detailed fragmentation pathways and diagnostic fragment ions. Methods: In the present work, the fragmentation pathways of five sulfonamides antibiotics, namely sulfamerazine, sulfathiazole, sulfadiazine, sulfadimethoxine and sulfamethoxazole, by High-Resolution Mass Spectrometry (HRMS) are presented. The HRMS spectra were recorded with a Q-TOF (Time of Flight) spectrometer with Electrospray Ionization (ESI) in both negative and positive mode. Results: Specific characteristic ions for each one of the sulfonamide antibiotics under positive ESI mode are proposed for the first time. Fragment ions of this particular class of analytes may be used to rapidly identify compounds with common structural features. Conclusion: The direct infusion of plasma samples, avoiding any prior chromatographic steps, to identify the existence of sulfonamide antibiotics is demonstrated herein.

Background: Phyllanthus urinaria, a traditional herbal medicine, has aroused widespread concern at home and abroad. However, there are few studies on the effects of Phyllanthus urinaria on CYP450. Therefore, this study aims to explore the main chemical compositions of Phyllanthus urinaria and its effect on the activity of CYP450 enzyme in rats. Methods: Acetonitrile and 0.1% Trifluoroacetic Acid (TFA) were used as mobile phase, along with the application of gradient elution to simultaneously determine the main chemical constituents in Phyllanthus urinaria by HPLC (r2>0.999). Sprague-Dawley (SD) rats, randomly divided into control group, low-dose group and high-dose group, were treated with normal saline and different doses of Phyllanthus urinaria extract solution, respectively. Additionally, the rats were given intragastric administration of cocktail probe (specific substrates of CYP450 isoenzyme) at 15th day; the plasma was collected by tail vein at various times. Furthermore, the UPLC-MS/MS method (r2>0.99) was used to detect the probe concentration, along with the evaluation of the activity of CYP450 enzyme according to the pharmacokinetic parameters of the probe. Results: Gallic acid, 3, 4-dihydroxybenzoic acid, caffeic acid, corilagin and ellagic acid were found in the Phyllanthus urinaria extract solution by HPLC. Compared with the control group, the metabolism of bupropion, metoprolol, midazolam and tolbutamide slowed down significantly in the Phyllanthus urinaria group, with no significant metabolic changes in phenacetin. Conclusion: Phyllanthus urinaria could induce activity of CYP2D6, CYP2B1, CYP3A4 and CYP2C9, without exerting a significant effect on CYP1A2.

Background: Enabling formulations have been implemented by the pharmaceutical industry as an effective tool for keeping Active Pharmaceutical Ingredient (API) in an amorphous state. Upon dosing in the amorphous state, many drugs which fail to demonstrate bioactivity due to the limited solubility and bioavailability of their crystalline form become bioavailable. Purpose: The analytical techniques use today for crystallinity detection are challenged by the sensitivity and robustness needed to achieve a 5% quantitation limit in low dose drug products. Our laboratory has developed a novel procedure capable of meeting this sensitivity and selectivity requirement. This is achieved by exploiting the differences in kinetic solubility of the formulated amorphous and free crystalline forms of API currently being used in dosage form platforms. Methods: Representative amorphous drug formulations were prepared and spiked with varying levels of crystalline drug substances to evaluate the selectivity and recovery of the crystalline drug substance from the product formulation. Kinetic solubility testing using a (i) Particle wetting phase, (ii) Particle suspending/erosion phase, (iii) Sampling time point and (iv) A total recovery determination for the drug substance. Results: The method selectively and quantitatively distinguishes crystalline drug substance from amorphous drug substance for samples spiked from 2.5% to 10% of the nominal label concentration of the API in the dosage form matrix. Conclusion: The kinetic solubility approach reported here achieves sensitive crystallinity quantitation for low drug level amorphous drug formulations at levels not yet achieved by complimentary analytical techniques.

Background: Tetralone derivatives, important resources for the development of new drugs which can act in the treatment of central nervous system disorders or participate in synthesis reaction for the synthesis of various pharmaceuticals, have great research value and a bright prospect in exploitation. Methods: A novel chiral HPLC method for efficient enantioseparation of eight tetralone derivative enantiomers was developed on cellulose based CHIRALPAK IC chiral stationary phase under normal mode by investigating the effects of type and content of organic modifier, column temperature and flow rate on retention and enantioselectivity. Besides, the specificity, linearity, stability, precision, accuracy and robustness of this method were also validated. Results: Satisfactory enantioseparation was obtained for all enantiomers in n-hexane/2-propanol mobile phase system at ambient temperature. The thermodynamic study indicated that the solute transfer from the mobile to stationary phase was enthalpically favorable, and the process of enantioseparation was mainly enthalpy controlled. This method met the requirements for quantitative determination of tetralone derivative enantiomers. Conclusion: This study can provide great and important application value for enantioseparation of eight pairs of newly synthesized tetralone derivative enantiomers under normal mode using CHIRALPAK IC chiral column.

Background: The decoction of Alisma orientale is used as a traditional medicine for the treatment of hyperlipidemia in China with a long clinical history. The present study undertook a detailed investigation to compare the hypolipidemic effect and chemical composition of two extracts of Alisma orientale prepared by boiling water and organic reagent, respectively. Methods: The hyperlipidemic mice were induced by administration of a High-Fat Diet (HFD) for one month. The body weight of mice and the serum Cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), Superoxide Dismutase (SOD), malondialdehyde (MDA), aspartate Aminotransferase (AST) and alanine aminotransferase (ALT) levels were determined. Meanwhile, the chemical constituents of the extracts were characterized using liquid chromatography-quadrupole time of flight mass spectrometry (LC-QTOF-MS/MS). Results: The better hypolipidemic effect was observed in mice administered with the decoction compared to that of methanol extract. In addition, the liver protective effects were confirmed by the photographs stained with oil red lipid stain. As for the chemical constituents in the extracts, twenty major components were identified or characterized using LC-QTOF-MS/MS. Among them, eight oligopeptides were characterized for the first time and proved to only exist in the decoction of Alisma orientale. Conclusions: It is reasonable to assume that the oligopeptides may partially contribute to the hypolipidemic effect of Alisma orientale. The structural characterization procedures also provide a suitable method to analyze oligopeptide analogues in plant samples.

Introduction: Wutou decoction has been wildly applied for the treatment of RA in China for several thousand years. Methods: This study aims to develop a highly sensitive and specific ultra performance liquid chromatography coupled with tandem mass spectrometry and electrospray ionization (UPLC-ESI-MS/MS) method to explore the pharmacokinetic properties of three representative bioactive alkaloids after intragastric administration of Wutou decoction in rats. Chromatographic separation was performed on a C18 column under the Multiple Reaction Monitoring (MRM) in the positive electrospray ionization (ESI) mode. The pharmacokinetic parameters were evaluated by software DAS 3. 0. Results: The validation of the method was achieved in accordance with the FDA guidelines. The results of pharmacokinetic study showed that the in vivo concentrations of benzoylmesaconine and benzoylhypaconine were significantly higher than benzoylaconine. Our PK results showed that these three compounds were quickly absorbed after the administration of Wutou decoction, and Tmax ranged from 30 min to 45 min. Conclusion: The results of pharmacokinetic study showed that the in vivo concentrations of benzoylmesaconine and benzoylhypaconine were significantly higher than benzoylaconine. There were also similar pharmacokinetic behaviors observed among BAC, BHA, and BMA after oral administration of WTD.

Background: A combination of alogliptin and pioglitazone is well tolerated. It does not increase the risk of hypoglycemia. In order to study the bioavailability of aloglipitn in the presence of pioglitazone, it is essential to have a method that can simultaneously detect both in human plasma. A protein precipitation-based method was used to determine alogliptin and pioglitazone simultaneously in human plasma. Protein precipitation causes ion suppression or enhancement in detection methods when compared to other methods. Objective: To simultaneously quantify alogliptin and pioglitazone in human plasma by LC-MS/MS based method. Methods: LC-MS/MS method for the simultaneous determination of pioglitazone and alogliptin in human plasma using stable isotope labelled compounds internal standards. The simple and one step solid phase extraction (SPE) was employed to extract the analytes from plasma. The extracted samples were separated on a C18 column by using a 25:75 (v/v) mixture of acetonitrile and 5 mM ammonium formate as the mobile phase at a flow rate of 0.5 mL/min. Results: The calibration curves obtained were linear (r2= 0.99) over the concentration range of 12.0- 2438.0 ng/mL for pioglitazone and 1.0-202.0 ng/mL for alogliptin. The results of the intra- and interday precision and accuracy studies were found to be within the acceptable limits. The analytes were stable under different stability conditions. All the validation results were found to be within the acceptable limits. The total analytical run time was 3.0 min. There was no interference from plasma matrices. Conclusion: The developed method is precise and adequately sensitive for detection and quantification of analytes. Thus, the method can be useful for bioavailability and bioequivalence (BA/BE) studies and routine therapeutic drug monitoring with the desired precision and accuracy.

Background: Thymol and carvacrol are the most important dietary constituents in thyme species. These two active compounds are used for the standardization of pharmaceutical compounds. Objective: In this work, a simple and reliable ultrasonic assisted dispersive solid phase microextraction method (USA-DSPME) coupled with high performance liquid chromatography-ultra violet detection system was developed to determine thymol and carvacrol in pharmaceutical syrups. The efficiency of SPME sorbent was examined through several sorbents and finally Graphene Oxide (GO) was applied for extraction of the analytes. Method: The efficiency of GO was compared with three reduced forms of GO adsorbents as well. Several effective factors on the extraction performance were investigated. Results: Under the optimized conditions for the GO sorbent, inter and intra-day relative standard deviations (RSDs, n = 3) and the Limits of Detections (LODs) were lower than 5.0% and 0.02 μg/ml, respectively. Moreover, good linear ranges were observed in wide concentration ranges with R-squared larger than 0.9961 for both thymol and carvacrol. Conclusion: The present method is reliable and simple for determination of carvacrol and thymol in pharmaceutical products.

Introduction: Acarbose, an anti-diabetic drug, is commonly used to treat diabetes mellitus type 2. Determinationof acarbose is crucial for routine quality control of acarbose tablets. Materials and Methods: In this report, a rapid, stable and precise method was developed and validated for the quantification of acarbose in tablets by 1H NMR. Two characteristic signals at 5.80 and 2.31 ppm were used to determine acarbose. The assay was linear over a concentration range of 0.25-10.0 mg/mL. The precision was 0.26% and 1.02% for signals at 5.80 and 2.31 ppm, respectively. The average recoveries of acarbose were 99.7% and 99.2%, with RSD values 0.51% and 0.61% at two signals, respectively. Results and Discussion: The content of tested tablets was 100.4% and 100.8% of the label claim with RSD values 0.96% and 1.47% at two signals, which met the requirement of Chinese Pharmacopoeia criteria for content uniformity of tablets. The assay has been successfully applied to determine the content of acarbose in tablets for quality evaluation. Conclusion: This method has been successfully applied to determine the content of acarbose in tablets for quality evaluation.

Objective: In this study, it was aimed to prepare an electrochemical sensor capable of assigning Norepinephrine in the presence of an interference such as ascorbic acid. Methods: A sensitive modified sensor was prepared by electrodeposition of p-aminobenzenesulfonic acid (p-ABSA) to the glassy carbon electrode by cyclic voltammetry. The electrooxidation of Norepinephrine was accomplished by cyclic and differential pulse voltammetry. Results: The current values were enhanced and the peak potentials of Norepinephrine and ascorbic acid were separated at the sensor compared to the bare electrode. There was linearity between the oxidation current and concentration of Norepinephrine ranging from 0.5 to 99.8 μM in phosphate buffer solution at pH 7.0. The limit of detection was 10.0 nM and the sensitivity was 0.455 μA/μM. Conclusion: The determination of Norepinephrine was successfully performed in real samples such as blood serum and urine at the poly (p-ABSA) sensor. To the best of our knowledge, this is the first study to detect Norepinephrine in the presence of ascorbic acid at poly (p-ABSA) modified sensor in the literature.

Objective: The aim of this study is to investigate the pharmacokinetics of benzoic acid, 4- [[(2-hydroxyethyl)amino]carbonyl], methyl ester in rats after intravenous and oral administrations at doses of 3 mL/kg. Methods: A rapid, high selective ultra-high performance liquid chromatographic electrospray quadrupole- time of flight mass spectrometry (UHPLC-ESI-Q-TOF/MS) method was applied to investigate the pharmacokinetics of benzoic acid, 4-[[(2-hydroxyethyl)amino]carbonyl]-, methyl ester with p-coumaric acid as internal standard (IS) in rats after intravenously and orally dosed. Results: The pharmacokinetic data of benzoic acid, 4-[[(2-hydroxyethyl)amino]carbonyl]-, methyl ester was analyzed in the two-compartment open model. The main pharmacokinetic parameters were, respectively, 36.474 μg·h/mL, 12.59 μg·h/mL (AUC0→∞), and T1/2α was 0.14 h, 0.359 h; T1/2β was 3.046 h, 5.646 h after intravenous and oral administrations. Conclusion: Benzoic acid, 4-[[(2-hydroxyethyl)amino] carbonyl]-, methyl ester was rapidly distributed in rat’s plasma with the absolute bioavailability of 34.5%.

Background: Biogenic Amines (BAs) are biologically active nitrogenous organic compounds of low molecular weight, which are frequently found in a wide variety of foods, beverages and herbs due to their toxic potential in humans. Male Silkworm Moth (MSM), a Traditional Chinese Medicine (TCM), has been exploited and utilized as nutritious liquor based on its traditional effects in the Chinese community. Objective: The objective of this study was to develop an HPLC with Dns-Cl derivatization method for characterizing overall BAs in MSM and providing data for further evaluating its activities and safety profiles. Methods: The method has acceptable sensitivity, precision, accuracy, selectivity and recovery, and was successfully applied to the determination of the BAs contents in MSM for the first time. Results: In the analysis of 10 batches of MSM samples, serotonin and dopamine were not found in detectable concentrations in any samples, and the most abundant amine found was putrescine. The mean values of tryptamine, phenylethylamine, putrescine, cadaverine, histamine, tyramine, spermidine, and spermine determined in the samples were found to be 34.7 mg/ kg, 16.1 mg/ kg, 218.3 mg/ kg, 37.9 mg/ kg, 12.1 mg/ kg, 18.2 mg/ kg, 4.5mg/ kg, and 0.9 mg/ kg, respectively. Conclusion: The contents of BAs in 10 batches of MSM were below the maximum recommended limits, and MSM can be used safely.

Background: Alendronate sodium is a common clinical osteoporosis drug for postmenopausal women; its determination is very important. However, there is no absorption of chromophores or fluorophores in the molecule, therefore, their direct determination is a challenge. Thus, establishing a common and direct method is very inspiring. Methods: According to the direct determination of alendronate sodium through the formation of a complex between alendronate sodium and divalent copper ion by capillary electrophoresis with ultraviolet detection, the dissolution profile of alendronate sodium tablet was established. The dissolution curves obtained from high-performance liquid chromatography method involving derivatization with 9- fluorenyl methylchloroformate and capillary electrophoresis with ultraviolet detector were found to be highly similar. Underivatized alendronate sodium can be determined by the capillary electrophoresis method. Results: Optimum conditions were as follows: background electrolyte including 25 mM CuSO4 at pH 4.59, 5 s injection time, 18 kV applied voltage, and 240 nm detected wavelength. Method validation indicated good linearity (r2>0.9993), precision of migration time with a relative standard deviation <1.5 % for intra-day and <3.6 % for inter-day, precision of peak areas <2.3 % for intra-day and <5.0 % for inter-day, limits of detection (0.01 μg/mL), limit of quantification (0.04 μg/mL) and recovery (90.6 %- 109.0 %). Conclusion: The proposed capillary electrophoresis method has been proved to be simpler, faster and more convenient to test dissolution profile of alendronate sodium tablet than that of high performance liquid chromatography.

Background: Liposomes continue to play an important role in drug delivery research due to their ability to improve transport and targeting of a wide range of active molecules. Analysis of liposomal components is a key point in the characterization and evaluation of formulation stability. The aim of this work was to develop and validate an HPLC-ELSD method for the characterization and quality control of liposomes. Methods: HPLC-ELSD method was validated by assessing selectivity, linearity, precision, accuracy, limit of detection and quantitation. The mobile phase consisted of a 0.1% (v/v) of trifluoroacetic acid (TFA) and methanol in gradient elution. Initial rate was 20:80 (0.1% TFA: methanol), with a ramp reaching 100% methanol. HPLC-MS/MS was used to confirm the presence of the fatty acid mixture in the analyzed lipids, as well as sub-products generated under pre-determined conditions in the stability study. Results: A HPLC-ELSD method has been developed to detect and measure cholesterol, phosphatidylcholine and lysophosphatidylcholine. High specificity, sensitivity and linearity within the predetermined range for all the compounds analyzed (R2>0.99) were obtained. Accuracy and precision results for all the compounds were within the acceptance limit of ≤5% and 90-110%, respectively. Mass spectrometry results showed complementary information about the phospholipid composition to evaluate the degree of degradation of liposomes over different storage conditions. Conclusion: The method was successfully applied as a quality control tool for the analysis of a wide range of lipids, present in liposomal formulations. HPLC-MS/MS was used to ensure complete elucidation of the lipid components and the detected lyso-forms.