Current Pharmaceutical Analysis - Volume 14, Issue 5, 2018

Background: Mesenchymal Stem Cells (MSCs) are multipotent stem cells with therapeutic potential in advanced therapies and regenerative medicine. Currently, MSCs are being widely studied as cellular medicine. In the design and development of medicines with cells, the study of viability is an important quality control requirement. Objective: Description of the main analytical techniques to study the MSCs viability as cellular medicine. Results: The cellular medicine must contain viable cells, for its use in vivo the cell viability must be at least greater or equal to 80%. Therefore, the study of cell viability of MSCs during its ex vivo expansion and its final formulation is a prerequisite before being administered. Conclusion: In this review the main analytical techniques based on studies of the cell membrane integrity, morphological assays and fluorescence biosensors are described. All of them are approved for use in quality control programs for clinical use of MSCs.

Background: Cabergoline, used for treating hyperprolactinemia, is hard to detect and determine in biological samples due to its dosage form. Materials and Methods: The trend hollow fiber mediating technique was used for preconcentration and extraction of cabergoline using the solvent bar and HPLC-UV. The parameter affecting the efficiency of this technique was optimized. The pH gradient was adjusted in accordance with the pKa of cabergoline. Other factors such as extraction time, stirring rate, the organic solvent, and salt addition were optimized based on the highest area obtained from HPLC-UV. Results and Discussion: Cabergoline was determined with a good correlation coefficient (r2 = 0.989), within the linearity range. 0.02ng mL-1 was the limit of detection for cabergoline and the Intra- and inter-day relative standard deviations were no more than 3.2% and 4.6%, respectively. This protocol offered a simple, sensitive, cost effective, and green method with great extraction efficiency for the detection and determination of cabergoline. Conclusion: Eventually, the presented method was proved applicable for human and urine samples.

Background: Acidic polysaccharides usually possess favorable bioactivities, and some of them are applied in medicines and dietary supplements together. However, it is a challenge to quantify these polysaccharides simultaneously. Methods: A method to Quantify Hyaluronic Acid (HA), Chondroitin Sulfate (CS) and a nonglycosaminoglycan acidic polysaccharide named AGSP was established by determining the contents of their disaccharides produced from the acid hydrolysis using hydrophilic interaction chromatographytandem triple quadrupole mass spectrometry in the positive mode. Results: This method had excellent linearities in a concentration range of 0.02~2.00 mg/mL for these standard acidic polysaccharides, the recoveries were all 95%~112%, and relative standard deviations (RSDs) were less than 5%. Limit of Detection (LOD) and Limit of Quantification (LOQ) ranged from 0.1 µg/mL to 4.1 µg/mL. It could be inferred that the demonstrated method was feasible to quantitatively detect uronic acid-containing polysaccharides. Conclusion: This method could be a useful tool for quality control of medicines and dietary supplements containing these acidic polysaccharides.

Introduction: The acute treatment of migraine headaches is one of the major fields in which rizatriptan is used. Materials and Methods: To determine rizatriptan in biological fluids, many analytical approaches have been reported based on Liquid Chromatography/ Tandem Mass Spectrometry (LC-MS/MS), High- Performance Liquid Chromatography (HPLC), Reversed Phase Liquid Chromatography with Diode Array Detection (RP-LC-DAD), and High-Performance Thin-Layer Chromatography (HPTLC). In this work, for the first time, Solvent Bar Microextraction (SBME) technique followed by HPLC with Ultraviolet (UV) was used to extract, detect, and determine rizatriptan in biological samples. The chemometric method was applied to optimize effects of various parameters affecting the extraction efficiency of the proposed method. Next, the extraction parameters including organic membrane solvent, extraction time, pH of donor and acceptor phases, salt addition, stirring rate, and sample solution temperature were optimized. The organic liquid membrane consisted of 1-Octanol immobilized in hollow fiber wall pores. A pH gradient was applied to migrate analytes from alkaline sample solution having a pH of 9.0, through the organic liquid membrane, into a solution of acidic acceptor having a pH of 2.9, existing inside the lumen of the hollow fiber. Having optimized the experimental conditions, extraction recoveries above 86% were achieved in various biological matrices. Result: As a result, pre-concentration factors above 127, acceptable Relative Standard Deviations (RSD%) with intra-day being 3.1% and inter-day being 4.6% were observed. The method showed decent linearity with estimation of coefficient above 0.999. Ultimately, it was used to detect and determine rizatriptan in human plasma and urine sample.

Background: Two sensitive and accurate methods have been developed for the determination of ceftriaxone sodium and sulbactam sodium in presence of their acidic, alkaline, oxidative, neutral and photolytic degradation products by chromatography including TLC and HPLC methods, as stability- indicating techniques. Methods: The first one is RP-HPLC method based on isocratic elution of the two drugs with their degradation products using mobile phase consisting of 0.01 M potassium dihydrogen phosphate buffer (pH 4.6) – acetonitrile (94 : 6, v/v) with UV detection at 220 nm. In the second method, densitometric separation of the binary mixture of ceftriaxone, sulbactam and their degradation products was achieved using a developing system consisting of dichloromethane : methanol : isopropanol : n-butanol : ammonia 33 %: water (22.5 : 22.5 : 20 : 5 : 5 : 2.5, by volume) with ultra violet detection at 270 nm. The proposed methods were validated with respect to ICH guidelines and the structures of all degradation products of both drugs were elucidated for the first time by Fourier Transformed Infrared Spectroscopy (FTIR) and mass spectrometry. Results: Good recoveries 100.10 ± 0.14 and 100.03 ± 0.68 for ceftriaxone and 99.19 ± 0.33 and 100.78 ± 0.95 for sulbactam were obtained using HPLC and TLC methods, respectively. The results obtained by applying the proposed methods were statistically analyzed and compared with those obtained by the reported method and no significant differences were found. Conclusion: The proposed methods were successfully applied for the determination of ceftriaxone and sulbactam in pure form, in pharmaceutical preparations and in presence of their degradation products.

Background: This study investigates fatty acids and sterol contents of some seeds used in Asian nutrition culture to prepare functional beverages, foods or fatty acids and sterols source. Objective: Current study presents an overview about some seeds as a valuable source for fatty acids and sterols extraction. Methods: Gas chromatography-mass spectrometry was employed to quantify fatty acids and sterol contents and compare them to standard components. Different ash values, free amino acids, and soluble sugars were investigated. High-Performance Thin Layer Chromatography (HPTLC) was employed to detect the components. Inter- and intra-day variations, linearity of the calibration curves, and the CV of accuracy for fatty acids and sterols were generally within the acceptable ranges. Results: The total oil content of the seeds ranged from 0.04% to 7.39%, with blue skullcap seed yielding the highest percentage of oil. The stigmasterol and β-sitosterol content of the oils ranged from 1.47 ± 0.03mg/100 g (canary grass seed oil) to 26.20 ± 0.40 mg/100 g (quince seed oil). The major monounsaturated fatty acid (MUFA)-oleic acid-was present in Quercus brantii oil, canary grass, and Crataegus aronia seeds. Linoleic acid was the most abundant polyunsaturated fatty acid (PUFA) in Entada rheedii (60.65 ±0.84%), canary grass (64.43±0.17%), and Cydonia oblonga seed oil (63.55 ±1.30%). Linolenic acid was the major fatty acid in the oil of blue skullcap (90.24 ±0.17%) and Lallemantia royleana seeds (85.18 ± 3.79%). Conclusion: Most of the detected seeds, especially Scutellaria lateriflora and L. royleana seeds were rich sources of phytosterols and essential fatty acids.

Background: Sitagliptin (SITA), is an oral anti-diabetic drug of dipeptidyl peptidase-4 (DPP-4) inhibitor class used as a monotherapy or in association with metformin to treat type II Diabetes Mellitus. Methods: Two new, sensitive and precise spectrofluorimetric methods have been developed for the determination of SITA in tablets and spiked human urine samples. SITA has a native fluorescence that was enhanced by 2 folds after addition of β-cyclodextrin (method A). The fluoregenic product was measured at 296 nm after excitation at 267 nm. In addition, SITA was derivatized by 7-chloro-4- nitrobenzofurazon (NBD-Cl) to a highly sensitive fluoregenic product was measured at 532 nm after excitation at 466 nm (method B). Results: The fluorescence intensities were directly proportional to the concentration over the range 0.35-10 μg mL-1 and 0.1-3 μg mL-1 for method A and B, respectively. The %RSD for inter-day and intra-day precisions was in a range of 0.16-0.92% and the accuracy results were in the range of 96.3% - 100.0%. Conclusion: Successful applications of the developed methods, for drug determination in pharmaceutical tablet and spiked human urine samples, were performed with high accuracy and precision.

Background: Many methods have been reported for the determination of ceftazidime such as liquid chromatography, spectrophotometry and electrochemical methods. Among these methods, electrochemical methods have their unique advantages such as rapidity, low cost and simplicity. Methods: In this work, electrochemical technique was used for the determination of ceftazidime using a carbon nanotube modified electrode. Sodium dodecyl benzene sulphonate (SDBS) was used to enhance the electrochemical response. Experimental parameters including accumulation time, concentration of SDBS and solution pH, were optimized for ceftazidime detection. Results: The carbon nanotube showed good electrocatalytic activity to the ceftazidime oxidation. The peak current of ceftazidime was greatly increased by SDBS. The detection linear range of this method was 2 to 55 μM and the detection limit was 0.11 μM. Conclusion: The modified electrode in this work was simple to fabricate and it was used for the determination of ceftazidime in ceftazidime vial.

Background: Traditional Chinese medicine has been widely used in China for a long time. As a new type of formulation, Traditional Chinese Medicine Injection (TCMI) is diffusely used clinically as a promising therapy for a variety of diseases in primary health care institutions of China, which has many advantages, such as functional reliability and speed of action. In particular, TCMI is very suitable for rescue in an emergency. Objective: The quantitation of gallic acid and bioactive components in Xuebijing injection. Methods: A rapid loop-based heart-cutting two-dimensional liquid chromatography method has been established and applied to the quantification of gallic acid and bioactive components in Xuebijing injection. Results: The method provided powerful separation and its performance has been verified by evaluation of transference (≥86.48%), accuracy (relative standard deviation ≤ 4.67%), recovery (≥95.44%), linearity (r2 ≥0.9993), and limit of quantification (≤ 1.66 μg/mL). Six compounds, gallic acid, hydroxysafflor yellow (HSYA A), ferulic acid, paeoniflorin, salvianolic acid B, and kaempferide, were identified with concentrations ranging from 0.35–1795.40 μg/mL in Xuebijing injection. Conclusion: The loop-based heart-cutting 2D-LC method established in this study may provide convenient and feasible approach for quality control of preparation and use of TCMI.

Background: The objective of present research work was to develop and validate rapid UHPLC method with short run time for the detection and quantification of recombinant human insulin content in microsphere formulations. Validation emphasizes mainly on studying the effects of the flow rate, wavelengths, column temperature, pH and organic content of the mobile phase and of presence of other additives on the method's accuracy. Methods: Chromatographic separation was achieved using YMC Triart C-18 column and the mobile phase was composed of the mixture (aqueous solution of 0.2M anhydrous sodium sulphate, pH 2.3 and acetonitrile) in a ratio 73:27 (%v/v). The column temperature and flow rate was 40ºC and 0.613 mL/min respectively whereas, UV detection was performed at 214 nm. All the method procedures were validated according to international conference on harmonization (ICH) guidelines. The retention time of recombinant human insulin was about 3.0 min with very short run time of just 5 min. Fluctuations in analytical conditions or presence of additives and impurities or insulin related analogs did not show any significant effect on the specificity and accuracy of the method. Results: The assay is reproducible with acceptable %RSD value (0.87%). Linearity was confirmed by correlation coefficient of 0.9999 over the range of 26.84-81.23 μg/mL. The accuracy of the method and % recovery of insulin content were found to be in between 98.02 and 99.71% while RSD ranged from 0.01 to 0.88%. The specificity of the method proved that no interferences occurred with RT of insulin peak. Conclusion: This new short validated method can be applied during development studies in research laboratories for high throughput testing as well as for quality control procedures of lot release testing, in-process quality control and finished product analysis.

Background: Estradiol valerate tablet is used for ovarian dysfunction, menopause, menopausal syndrome, breast and prostate cancer. Introduction: HPLC method coupled with photodiode-array detection has been used to develop a simple and sensitive reversed-phase method for quantitative determination of all possible related impurities in Estradiol valerate tablet. Methods: Zorbax eclipse XDB C8(150mmx4.6mm,5μm) column was used. The mobile phase was gradient prepared from 30:10(v/v) acetonitrile-methanol (component A) and water (component B); the gradient program (time (min)% B) was 0.01/40, 40.0/40, 40.1/15, 50.0/15, 50.1/40, 55.0/40, 55.1/40, 60.0/40. Eluting compounds were monitored at UV detection (220nm) and fluorescence detection (Ex:280nm, Em 310nm). Result: All standard curves obtained exhibited good linear regression (r0.9996) within the tested range. All the average recovery rates were in the range of 99.2%-100.8% and RSD were less than 2.0%. The method was successfully validated according to ICH guidelines acceptance criteria for robustness, selectivity, linearity, accuracy and precision. Conclusion: Estradiol valerate was subjected to oxidative, acid, base, thermal and photolytic stress, and analysis was conducted to determine the amounts of related impurities.

Background: Cetuximab (CET) is a monoclonal antibody that inhibits Epidermal Growth Factor Receptor (EGFR) used for immunotherapy of different types of cancer. Additionally, CET used in combination therapy, potentiates the effects of chemotherapy and radiation therapy in eradicating well-established tumors. Recently, a combination of CET and newly developed chemotherapeutic candidate drugs are being investigated for use as new-generation chemotherapy. Introduction: Due to the increasing importance of this class of biopharmaceuticals, our dynamic field of research has arisen around the establishment of an immunoassay method with a highly specific and sensitive to quantify CET at low concentration levels in human plasma. This study describes the development and full validation of a new enzyme-linked immunosorbent assay (ELISA) with high sensitivity and selectivity for bioanalysis of CET. Methods: The assay involved the non-competitive binding reaction of CET to its specific antigen (human epidermal growth factor protein; EGFR) followed by a chromogenic enzyme reaction for immune detection of the CET- EGFR complex and color development. Technically, CET was captured by EGFR antigen protein immobilized onto a 96-well assay plate. The CET- EGFR complex formed onto the plate wells was quantified, for the first time, using alkaline phosphatase enzyme labeled anti-human IgG (ALP-IgG) and para-nitrophenyl phosphate substrate (pNPP) as a chromogenic substrate for alkaline phosphatase enzyme. Results: The optimum conditions for conducting the proposed ELISA were established and its analytical performance was evaluated as per the guidelines for the validation of immunoassays for bioanalysis. The assay Limit Of Detection (LOD) was 1.5 ng/mL and the effective working dynamic range was 5- 6250 ng/mL. The accuracy and precision of the assay were proved. Conclusion: We have developed and validated a highly sensitive and selective ELISA for quantitation of CET in plasma samples. The CET- EGFR complex formed onto the plate wells were quantified, for the first time, using alkaline phosphatase enzyme labeled anti-human IgG (ALP-IgG) and paranitrophenyl phosphate substrate (pNPP) as a chromogenic substrate for alkaline phosphatase enzyme. An analyst can process a batch of ~ 200 samples, in triplicate, per day. The proposed ELISA method for CET shows acceptable precision and, accuracy and adequate sensitivity to contribute to study its pharmacokinetic (PK), Pharmacodynamic (PD), Therapeutic Drug Monitoring (TDM) and appears to be suitable for employment in all clinical laboratories.