Current Pharmaceutical Analysis - Volume 16, Issue 3, 2020

Background: A sensitive, reliable liquid chromatography-tandem mass spectrometry (LCMS/ MS) method has been developed and applied to detect the evodiamine (EVO) in rat plasma after animals were given EVO directly. However there is almost no research on the detection of EVO after animals were given EVO-loaded solid lipid nanoparticles (EVO-SLN). Objective: In this study, a more sensitive and rapid modified LC-MS/MS method for the quantification of EVO in rat blood was developed and validated to evaluate the role of SLN in vivo. Methods: Plasma samples were taken from animals orally administered EVO-SLN or free EVO, proteins were extracted using diethyl ether containing the internal standards (IS) arbidol hydrochloride, and the mixture was fractionated by liquid chromatography. Quantitative detection of EVO was based on gradient elution in a mobile phase of acetonitrile-0.2% formic acid in water (70:30, v/v). Results: The calibration curve was linear (r2>0.999, n=9) over the concentration range from 0.1 to 250 ng/mL. Peaks in triple-quadrupole MS were detected for EVO at m/z 304.2→134.1 and for IS at m/z 479.1→343.0. Mean recovery of EVO was more than 93%. Intra and inter-day precision were within 2.7%. In pharmacokinetics studies, EVO-SLN exhibited much higher bioavailability and absorption than free EVO. Conclusion: The developed method in this work can provide a sensitive, effective and rapid process for the analysis of EVO in whole blood samples. The pharmacokinetics results suggest that the usefulness of SLN for improving oral bioavailability of poorly soluble drugs.

Background: Atorvastatin (AT) belongs to cholesterol-lowering agents, commonly used in patients with an increased risk of cardiovascular disease. The drug, as well as its hydroxyl metabolites, exhibit pharmacological activity, and their plasma levels may be helpful in the assessment of the therapeutic effectiveness. Objective: Development and validation of a fast and reproducible RP-HPLC method with UV detection for the simultaneous determination of atorvastatin and its active metabolites, para-hydroxy-atorvastatin (p-OH-AT) and ortho-hydroxy-atorvastatin (o-OH-AT) in human plasma. Methods: Optimal conditions of chromatographic separation of the analytes, as well as rosuvastatin, chosen as an internal standard, were studied. The absorbance of the compounds was measured at λ=248 nm. Validation of the method was performed. The usefulness of the method was confirmed for determination of the analytes in plasma of patients treated with the drug. Results: Total peak separation was achieved at LiChrospher 100 RP-18 column with a mobile phase composed of methanol and water (1:1,v:v) and a flow rate of 1.2 ml/min. The method was linear in the ranges of 0.025 - 1.0 μg/ml for AT, o-OH-AT and p-OH-AT. Intra- and inter-assay precision expressed as relative standard deviation was ≤13% for AT, ≤12% for p-OH-AT and ≤11% for o-OH-AT. Intraand inter-day accuracy of the method, expressed as a relative error, was ≤15%. Conclusion: The elaborated HPLC method is specific, repeatable, reproducible, adequately accurate and precise and fulfills the validation requirements for the bioanalytical method. The method was successfully applied for analysis of atorvastatin and its o-hydroxy metabolite in plasma of patients treated with the drug.

Background: Spectrophotometry and thin layer chromatography have been commonly applied in pharmaceutical analysis for many years due to low cost, simplicity and short time of execution. Moreover, the latest modifications including automation of those methods have made them very effective and easy to perform, therefore, the new UV- and derivative spectrophotometry as well as high performance thin layer chromatography UV-densitometric (HPTLC) methods for the routine estimation of amrinone and milrinone in pharmaceutical formulation have been developed and compared in this work since European Pharmacopoeia 9.0 has yet incorporated in an analytical monograph a method for quantification of those compounds. Methods: For the first method the best conditions for quantification were achieved by measuring the lengths between two extrema (peak-to-peak amplitudes) 252 and 277 nm in UV spectra of standard solutions of amrinone and a signal at 288 nm of the first derivative spectra of standard solutions of milrinone. The linearity between D252-277 signal and concentration of amironone and 1D288 signal of milrinone in the same range of 5.0-25.0 μg ml/ml in DMSO:methanol (1:3 v/v) solutions presents the square correlation coefficient (r2) of 0,9997 and 0.9991, respectively. The second method was founded on HPTLC on silica plates, 1,4-dioxane:hexane (100:1.5) as a mobile phase and densitometric scanning at 252 nm for amrinone and at 271 nm for milrinone. Results: The assays were linear over the concentration range of 0,25-5.0 μg per spot (r2=0,9959) and 0,25-10.0 μg per spot (r2=0,9970) for amrinone and milrinone, respectively. The mean recoveries percentage were 99.81 and 100,34 for amrinone as well as 99,58 and 99.46 for milrinone, obtained with spectrophotometry and HPTLC, respectively. Conclusion: The comparison between two elaborated methods leads to the conclusion that UV and derivative spectrophotometry is more precise and gives better recovery, and that is why it should be applied for routine estimation of amrinone and milrinone in bulk drug, pharmaceutical forms and for therapeutic monitoring of the drug.

Background: The ability of the normalized spectra when used as a divisor and in combination with isosbestic point to resolve complex binary or ternary mixtures, Candesartan and Hydrochlorthiazide binary mixture was taken as a model. Introduction: A green simple smart and accurate method using ethanol as a solvent namely simultaneous derivative ratio (SIDD) was applied to prove the power of normalized spectra and isosbestic point as spectrophotometric resolving tools. Methods: In the proposed SIDD method, the zero order spectra of drugs were simply manipulated using the normalized spectra of CAN as divisor to obtain the ratio and first derivative spectra in two successive steps. Firstly, the total amplitude at isosbestic point 255.4 nm of the ratio spectra of the mixture was measured representing the total actual concentration of both drugs in the mixture. Then, the first derivative of the ratio spectra was obtained to determine Hydrochlorothiazide concentration at 233 nm. While the concentration of Candesartan was determined subsequently by subtracting the Hydrochlorothiazide concentration calculated after derivatization from the total concentration of both drugs obtained at the ratio spectra before the derivatization step. Results: The SIDD was successfully applied for simultaneous determination of both drugs in their pure form or in their binary mixture either in synthetic prepared mixtures or in combined dosage form the adopted method was validated according to the ICH guidelines and the results were found to be within the acceptable limits. Conclusion: The adopted method highlighted the important role of normalized spectrum when used as a divisor in addition to the importance of isosbestic point to resolve severely overlapped spectra. All the measurements were carried using ethanol which is considered one of the greenest solvents making the method an environmentally friendly one. the adopted method was validated according to the ICH guidelines and the results were found to be within the acceptable limits.

Background: Hepatocellular carcinoma (HCC) is one of the most deadly liver malignancy found and Hepatitis C virus (HCV) is a prominent risk factor for this disease. Prognosis of HCC is poor; initiate the need of markers to discover therapeutic targets in HCC. Introduction: Clinical staging systems of HCC composed of tumor characteristics along with liver function test are important in prognosis but they are not precise. Molecular profiling can lead to a better understanding of the physiopathology of HCC and can help in the development of novel therapeutic approaches. Methods: 64 HCC serum samples (shifted from HCV) were graded into stage I- IV; along with +ive (3 Hepatitis C) and -ive control (2 healthy persons). Proteins were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and differential mRNA expression from serum samples of different HCC stages was confirmed by Real Time Polymerase Chain Reaction (qPCR). Results: HCC serum proteins displayed differential expression of glutathione s-transferase (GST), glypican-3 (GPC3), vitronectin (VTN), and clusterin (CLU) by SDS-PAGE. GST was expressed in -ive control, while GPC3 was found in both -ive and +ive control. The qPCR analysis, display more than 0.07 fold decrease in GST in I-IV HCC stages. The highest increase in HCC stages was observed by GPC3; about 4 fold increase in I-IV stages. VTN show 1.7-3.4 fold; while CLU show 2-3.5 fold increase in four stages of HCC. Conclusion: GPC3, VTN and CLU in combination can be good potential markers for differentiating stages (I-IV) of HCC.

Background: EAI045 is the fourth-generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR TKI), which can overcome acquired resistance to the third-generation EGFR TKIs and is the first allosteric inhibitor that targets T790M and C797S EGFR mutants. Methods: A rapid and sensitive LC-MS/MS method was established and validated for the quantification of EAI045 in rat plasma. Chromatographic separation was carried out at 25°C on a Hypersil GOLD C18 column (50 mm x 2.1 mm, 3 μm) and eluted on a gradient mobile phase of water (containing 0.1% formic acid) and acetonitrile at a flow rate of 0.5 mL/min. The mass spectrometer was operated in the positive ESI mode and selected reaction monitoring mode. Results: The assay was validated over a concentration range of 1.0 - 1000 ng/mL for EAI045 with a lower limit of quantification (LLOQ) of 1.0 ng/mL. The intra- and inter-batch accuracy for the EAI045 ranged from 92.25% to 97.18% and 95.94% to 102.69%, and the intra- and inter-batch precision for the EAI045 ranged from 1.41% to 4.57% and 5.18% to 6.37%, respectively. The extraction recovery, matrix effect and stability met all requirements of the guidelines for bioanalytical method validation. Conclusion: The rapid and sensitive LC-MS/MS method was successfully applied in a pharmacokinetic study of EAI045 following oral administration (5 mg/kg) to rats.

Background and Objective: Radix Salvia miltiorrhiza (RSM) has been used clinically for the prevention and treatment of cardiovascular diseases; therefore, it is important to strengthen its quality management. Considering multiple constituents when assessing RSM quality is essential. We established a simple, rapid method to identify and quantify the major bioactive constituents in RSM using ultra high performance liquid chromatography (UPLC) coupled to a triple quadruple mass spectrometry (QqQ-MS). Methods: We analyzed 17 markers from 50 batches of wild S. miltiorrhiza samples that were collected from different locations in China. The ultrasonic extracts of all samples were determined using the UPLC-QqQ-MS method and were assessed by hierarchical cluster analysis (HCA). Results: We used this method to analyze 50 sample batches of the 17 compounds and obtained results with excellent linearity (R2, 0.9915-0.9997), precision (relative standard deviation, RSD, 0.15-1.94%), repeatability (RSD, 1.28-4.71%), stability (RSD, 0.97-5.60%) and recovery (RSD, 0.305-6.40%). The hierarchical cluster analysis was used to classify the 50 samples based on the characteristics of the 17 compound markers. Conclusion: We demonstrated that the developed method was simple, reproducible and sensitive, and it is capable of systematic and scientific evaluation for quality control of RSM. The HCA clearly demonstated that the RSM samples from different locations were significantly different and the quality of wild Radix S. miltiorrhiza could generally be judged according to its geographical origin.

Background: The complexity of follicular fluid metabolome presents a huge challenge for qualitative and quantitative metabolite profiling and discovery of the comprehensive biomarkers. Objective: In order to address this challenge, novel SWATHtoMRM metabolomics method was used for providing broad coverage and excellent quantitative capability to discover the human follicular fluid metabolites related to age and evaluate their relationship with pregnancy outcome and oocyte senescence. Methods: The patients were divided into four groups according to age, including group A (28 cases, 21- 27 years old), group B (42 cases, 28-34 years old), group C (31 cases, 35-41 years old), and group D (24 cases, 42-48 years old). Follicular fluid samples from 125 IVF patients were analyzed. The differential ions among the four groups were identified by principal components analysis according to accurate mass, isotope ratio, and tandem mass spectroscopic spectra. Then, the differential metabolic pathways were further identified by a KEGG cluster analysis. Results: A total of 18 metabolites in the follicular fluid differed among the four groups, including amino acids, lipids, hormones, and vitamins. A total of 15 metabolites, including 6-oxohexanoate, phenylalanine, proline, hexadecanoic acid, linoleate, arachidonate, oleic acid, docosahexaenoic acid, LysoPC(16:1), LysoPC(20:5), LysoPC (20:3), 25-hydroxyvitamin D3, 5-dehydroepisterol, 27- hydroxycholesterol, and 5beta-cholestane-3alpha,7alpha,12alpha,23,25-pentol, were down-regulated with age and 3 metabolites, including LysoPC(18:3), LysoPC(18:1), and 13,14-dihydroretinol, were upregulated with age. Conclusion: Our study provides useful information for revealing the relationship between age and female reproductive capability.

Background: Scutellariae Radix (Huangqin) is commonly processed into 3 products for different clinical applications. However, a simple analytical method for quality control has rarely been reported to quickly estimate the degree of processing Huangqin or distinguish differently processed products or unqualified Huangqin products. Objective: To study a new strategy for quality control in the processing practice of Huangqin. Methods: Seven kinds of flavonoids that mainly exist in Huangqin were determined by HPLC-DAD. Chromatographic fingerprints were established to study the variation and discipline of the 3 processed products of Huangqin. PCA and OPLS-DA were used to classify differently processed products of Huangqin. Results: The results showed that baicalin and wogonoside were the main components in the crude and the alcohol Huangqin herb while baicalein and wogonin mainly existed in carbonized Huangqin. The results of mathematical statistics revealed that the processing techniques can make the quality of medicinal materials more uniform. Conclusion: This multivariate monitoring strategy is suitable for quality control in the processing of Huangqin.

Background: Itopride used for the gastrointestinal symptoms caused by reduced gastrointestinal mobility. For the first time rapid, low cost and green voltammetric method has been applied to analyze itopride in pharmaceutical formulation. Methods: Cyclic Voltammetry (CV), Linear Sweep Voltammetry (LSV), Square Wave Voltammetry (SWV) and Differential Pulse Voltammetry (DPV) methods have been applied in this study. Results: Na2SO4 (1M) supporting electrolyte exhibited sharper anodic peak current than other used supporting electrolytes; glassy carbon electrode (GC) working electrode shows better results than platinum electrode (Pt). SWV results show the lowest limit of detection and quantitation values of 2.3 and 18.1 μg.mL-1, respectively. SWV recovery is 100.56% and 100.46% for 50 μg.mL-1 and 100 μg.mL-1 of commercially available itopride tablets, respectively. Furthermore, SWV inter and intraday results precessions are better than other used methods with 0.96 and 0.56% RSD, respectively. Conclusion: The optimum method of applied methods in this study is SWV method. Voltammetry showed low LOD and LOQ values with high accuracy and precession in addition to comparable repeatability and reproducibility values.

Background: The HL60-IL6 assay has been initially established, but the process of the assay and calculation was not simplified. And there are no reports on whether it can be applied to detect pyrogen contamination in the monoclonal antibody. Objective: The study aimed to improve the HL60/IL-6 assay and detect the pyrogens in the monoclonal antibody drug by HL60-IL6 assay. Methods: The human promyelocytic leukemia cell line (HL-60) was incubated with pyrogen standard solution, such as lipopolysaccharide (LPS), zymosan and lipoteichoic acid (LTA),or monoclonal antibody sample solution for 48 hours, and then cytokines interleukin-6 (IL-6),secreted from HL-60, were measured by ELISA. The study further described the standard curves on OD (Optical Density) value of IL-6 responding to pyrogen stimulation, and determined the content of pyrogen in the monoclonal antibody production after validation. In addition, the sensitivity of HL60 to three pyrogens was evaluated to establish one standard curve to determine endotoxin and non-endotoxin level. Then, the credibility of standard curves was evaluated. After improvement of the assay, 9 monoclonal antibody batches were assayed for pyrogens in parallel with the Rabbit Pyrogen Test (RPT) and HL60/IL-6 assay. Results: It was achieved that the standard curve between OD value of IL-6 and pyrogen concentration was established. Then, it was found that the sensitivity of HL60 responding to LPS was the weakest, as a result of which, only LPS standard curve needs to be described in each test for detection of pyrogens. Besides, to evaluate the credibility of standard curve, the parameters of the standard curve were restricted and the resulting interpretation was also specified. 3 Bevacizumab batches failed the RPT, which also showed pyrogenic contamination by the HL60/IL-6 assay. Conclusion: HL60-IL6 assay was improved and can be applied to pyrogen detection of monoclonal antibody.

Objective: A validated liquid chromatography-tandem mass spectrometry method (LCMS/ MS) was established to simultaneously determine the concentration of triflusal and its main metabolite 2-hydroxy-4-trifluoromethyl benzoic acid(HTB) in human urine. Methods: The separation was performed on a Dikma C18 column using isocratic elution with acetonitrile-4 mmol/L ammonium acetate aqueous solution containing 0.3 % formic acid water (78: 28, V/V). The method involved extraction with methanol using protein precipitation. The precursor-toproduct ion transitions with multiple reaction monitoring was m/z 247.1→161.1, 204.8→106.7and 136.9→93.0 for triflusal, HTB and salicylic acid(IS), respectively. The method showed good linear relationships over the ranges of 0.08 to 48 μg/mL and0.5 to 50 μg/mL. Results: It was the first time that a urinary excretion study of triflusal capsule as oral. The cumulative urinary recovery showed 8.5% and 2.7% for triflusal and HTB, respectively. Conclusion: This method was successfully used for evaluating the pharmacokinetic properties of triflusal and HTB in urine in Chinese healthy subjects.