Current Pharmaceutical Analysis - Volume 18, Issue 2, 2022

In this review, numerous analytical methods to distinguish pigments in tattoo, paint, and ink items are discussed. The selection of a method was dependent upon the purpose, e.g., quantification or identification of pigments. The introductory part of this review focuses on describing the importance of setting up a pigment-associated safety profile. The formation of different degradation chemical substances as well as impurity trends can be indicated through the chemical investigation of pigments in tattoo products. It is noteworthy that pigment recognition in tattoo inks can work as a preliminary method to identify the pigments in a patient's tattoo before being removed by laser therapy. Contrary to the study of banned pigments, the identification process usually requires only a few indication substances for positive dyes. In general, tattoo pigments are almost insoluble in aqueous solutions, and many organic solvents and various pigment analyses have been conducted. It is proposed that in the future, laboratories concerned with tattoo substance analysis should have access to extensive pigment specifications and spectroscopic databases. The most important and recent physiological side effects of tattooing have been discussed in this review.

Background: The objective of this research was to screen metabolites with specificity differences in the lung tissue of paraquat-poisoned rats by metabolomics technology and chi-square test method, to provide a theoretical basis for the study of the mechanisms of paraquat poisoning, and to use machine learning technology to construct a paraquat poisoning diagnosis model. This provided an intelligent decision-making method for the diagnosis of paraquat poisoning. Methods: 18 paraquat-poisoned rats (36 mg/kg) and 16 positive control rats were selected. Lung tissue from each rat from both groups was extracted and analyzed by GC-MS. The chi-square test for feature evaluation was used to screen the difference in specific metabolites in the lung tissue between the paraquat-poisoned rats and the control group, and the SVM classification machine learning algorithm was used to construct an intelligent diagnosis model. Results: In the end, a total of 14 significant metabolic differences were identified between the two groups (P < 0.05). The sensitivity, specificity, and accuracy of the constructed SVM paraquat poisoning diagnostic model reached 95%, 95% and 96.67%, respectively. Conclusion: Based on metabolomics technology, the chi-square test for feature evaluation was used to successfully screen the changes of specific metabolites produced in the lungs after paraquat- poisoning, and the diagnosis model based on SVM was constructed to provide an intelligent decision for the diagnosis of paraquat poisoning.

Background and Objectives: SM-1 is a new synthetic small molecular compound with anti-tumor activity. The metabolism of SM-1 is a key parameter that needs to be evaluated to provide further insight into drug safety and efficacy in the early phases of drug development. Methods: In this study, the biotransformation process of SM-1, including the metabolic pathways and major metabolites, was investigated based on a liquid chromatography-mass spectrometry method. Upon incubation of SM-1 with human liver microsomes, five metabolites were identified, namely dihydrodiol formation (R1), hydroxylation (R2, R3, and R5), and debenzylation (R4) of SM-1, with R1 and R4 being the major metabolites. The enzyme kinetic parameters of SM-1 were determined by a liquid chromatography-tandem mass spectrometry method. The enzyme kinetics of SM-1 obeyed the Michaelis-Menten equation. The Vmax, Km, and CLint of SM-1 in HLMs were 14.5 nmol/mg protein/h, 6.32 μM, and 2.29 mL/mg protein/h, respectively. Results: The chemical inhibition studies showed that CYP450 isoenzymes were responsible for SM-1 metabolism in HLMs, and CYP3A4 was the major CYP450 isoenzyme involved in the metabolism of SM-1; these findings were confirmed by using the human recombinant CYP3A4. Conclusion: Through the identification of the biotransformation pathways and enzyme kinetics of SM-1, the metabolic enzymes for SM-1 in HLMs are characterized.

Background: Compound Niuhuang Xiaoyan capsule is a realgar and cinnabar-containing traditional Chinese medicine, the contents of total arsenic and total mercury are found over regulation defined limits. However, only small amounts of arsenic in realgar and mercury in cinnabar are dissolved in the gastric juice, so the determination of soluble arsenic and soluble mercury is more significant than that of total arsenic and total mercury in Compound Niuhuang Xiaoyan capsule. Objective: To study the contents of total arsenic, total mercury, soluble arsenic and soluble mercury in Compound Niuhuang Xiaoyan capsule. Methods: Microwave digestion and semi-bionic extraction were used to pretreat the samples of Compound Niuhuang Xiaoyan capsule, and the inductively coupled plasma mass spectrometry method was established to determine the contents of total arsenic, total mercury, soluble arsenic and soluble mercury in Compound Niuhuang Xiaoyan capsule. The accuracy of the established method was further evaluated by using a certified standard reference material prepared from dried citrus leaves (GBW10020(GSB-11)). Results: The spiked recoveries were within 95-105 %. The R.S.D% of repeatability (N=6) and precision (N = 6) were below 5.0 %. The correlation coefficients of linear (R) for arsenic and mercury were both above 0.998. The limits of quantification (LOQ) were below 0.1 μg/L. The contents of As and Hg were defined in the dried citrus leaves and very near to the standard values provided by the manufacturer. The established method was applied for the analysis of three batches of Compound Niuhuang Xiaoyan capsule produced by three different manufacturers successfully. Insufficient realgar or cinnabar was formulated in some Compound Niuhuang Xiaoyan capsule samples. Conclusion: The analysis showed that the contents of soluble arsenic and soluble mercury in artificial gastric juice were significantly less than that of total arsenic and total mercury in Compound Niuhuang Xiaoyan capsule. The results provided a reference for further study on the toxicology and pharmacokinetics of Compound Niuhuang Xiaoyan capsule.

Background: Cyclodextrins (CDs) are commonly used host molecules of inclusion complex. However, due to the lack of a sensitive determination method, the absorption process of CDs remains unclear. Objective: In this study, an oleuropein (OL) inclusion complex employing hydroxylpropyl-betacyclodextrin (HP-beta-CD) as host molecules was prepared and the formation of inclusion complex was ascertained by FT-IR and DSC. Spectrophotometry was established for the determination of HP-beta-CD, based on the fact that the absorbance of phenolphthalein (PP) decreased in the presence of HP-beta-CD. Methods: The assay conditions were optimized to augment the method sensitivity. Molecular docking was employed to verify the strong interaction between PP and HP-beta-CD. The permeation process of free HP-beta-CD, HP-beta-CD of OL inclusion complex, free OL, and OL in the inclusion complex, was examined using an in vitro mouse small intestine model. Results: Though HP-beta-CD possessed a hydrophilic outside shell, it could permeate through the mouse small intestine quickly with a cumulative permeating amount of over 90% in 2 h. Free HPbeta- CD, the host molecule HP-beta-CD, and guest molecule OL of the inclusion complex exhibited consistent permeating profiles across the mouse small intestine. Conclusion: The approach for the determination of HP-beta-CD was accurate and precise (%RSD=2.98).

Background: Omeprazole has poor water solubility, is unstable in acidic solutions and undergoes first-pass metabolism, which results in lowering its bioavailability. A solid Self-Nano Emulsifying Drug Delivery System (SNEDDS) was previously prepared to enhance its dissolution. Objective: Development and validation of an RP-HPLC method with UV detection for the determination of omeprazole in 0.1 N HCl and in 0.01 M phosphate buffer (pH 7.4). Methods: Validation was according to the ICH Q2 (R1) guidelines in terms of linearity, accuracy and precision, lower limit of quantification, sensitivity, specificity, and robustness. The developed and validated method was used to study the in-vitro dissolution of the drug from the solid- SNEDDS, commercial products, and the unprocessed drug. The dissolution was studied in 500 ml of 0.1N HCl during the first 2 hours, and 900 mL of 0.01 M phosphate buffer (pH 7.4) during the last hour (37 ± 0.5 °C and 100 rpm). Results: The method was linear in the range 1-50 μg/ml, accurate and precise as indicated by the ANOVA test. It was specific to the drug and the pharmaceutical excipients did not affect the determination of its concentration. The method was robust to small changes in pH, composition, and flow rate of the mobile phase. The dissolution rate of omeprazole from the Solid-SNEDDS was faster than that from two commercial dosage forms and than the dissolution rate of the unprocessed drug. Conclusion: The method met the acceptance criteria and was applied successfully in studying the rate of dissolution of the drug.

Background: Thiamine hydrochloride (THY), also known as vitamin B1, is a watersoluble vitamin usually found in food and considered as a dietary supplement and treatment in case of vitamin deficiency, for example, in malabsorption and chronic diarrhea. Objectives: Rapid, green, and sensitive methods for the assay of vitamin B1 (thiamine hydrochloride, THY) in pharmaceutical forms, using normal and reverse flow injection manifolds and sulfonamide drugs as green and safe reagents were reported. Sulfamethoxazole and sulfadimidine drugs were used rather than toxic and expensive reagents. Methods: nFIA and rFIA manifolds were used for automated the reaction of THY with diazotized sulfamethoxazole (DSMZ) and diazotized sulfadimidine (DSDM) in an alkaline medium, respectively. The absorbance of the resultant red-orange azo dyes was monitored spectrophotometrically at λmax494 and 496nm for both methods, respectively. Results: The linearity of the suggested methods was in the ranges of 2 till 80 μg/mL (LOD 0.69 μg/mL, %RSD 0.32, n=6) for nFIA method and 0.5-70μg/mL (LOD 0.29 μg/mL, %RSD 0.85, n=6) for rFIA. The sampling frequency was 84 and 51 injections per hour for nFIA and rFIA methods, respectively. Chemical and physical variables for both methods were studied carefully. Conclusion: Both flow injection modes were effectively applied in the assay of THY in its pharmaceutical forms. The results were compared with those of the standard pharmacopeia method, and the statistical analysis indicated insignificant differences in accuracy and precision between the methods.

Background: Extensive therapeutic drug monitoring needs an analytical method for efficient and sensitive quantification of analytes of interest in clinical pharmacology. Objectives: A rapid, robust, sensitive and simple UPLC-MS/MS method to quantify Methsuximide (Ms) and N-desmethyl methsuximide/Normesuximide (MsMET) in human plasma was optimized, developed, and validated for application in a pharmacokinetic study. Methods: Reverse phased chromatography was performed using Zorbax SB-C18, 4.6 x 75 mm., 3.5 μm as stationary phase, methanol and 0.1% formic acid (60:40 v/v) as mobile phase which was delivered isocratically at a flow rate of 0.9 mL/min. The sample injection volume was 5 μL. Mass spectrometric quantification of the analytes was performed using positive electrospray ionization as mass interface along with multiple reaction monitoring (MRM) as acquisition mode. Results: The selected mass transition ions for analyte, metabolite and its respective internal standards are as follows, precursor ion (m/z) and product ion (m/z): Ms (204.06 and 119.02), MsMET (190.05 and 119.82), Ms internal standard (MsIS) (209.17 and 124.02), and MsMET internal standard (MsMETIS) (195.09 and 124.16), respectively. The current method was found to be linear for Ms (60.720-6043.800 ng/mL) and MsMET (60.389 - 6010.800 ng/mL) with r2 values not less than 0.999. The mean recoveries of all analytes ranged between 71.37 and 86.38 percentage. Conclusion: This method was validated in accordance with USFDA’s bioanalytical guidelines. This method could be applied for a routine analysis of Ms and MsMET in clinical pharmacological practice.

Background: Design of experiment (DOE) is considered the most powerful tool to identify factors that affect variation and improve the response by tuning these factors. In the present work, DOE was applied to establish an innovative, sensitive, and precise HPLC method for the simultaneous determination of Escitalopram oxalate, Paroxetine hydrochloride hemihydrate and Clonazepam in the presence of their related compounds in drug substance and drug products. Methods: Buffer molarity, % organic modifier (acetonitrile content) at the beginning/end of the gradient, flow rate at the beginning/end of the gradient, pH of the mobile phase, and column temperature were screened using Plackett-Burman design (PBD) model. The main effect plot showed that % organic modifier at the beginning/end of gradient and flow rate at the beginning of gradient were statistically significant variables influencing peaks resolution (p<0.05). Box-Behnken design (BBD) was then used as an optimization model in order to achieve the highest possible resolution with the least possible experimental trials through studying the interaction and quadratic effects of these three factors. Finally, the optimum condition for predicated peak resolution could be achieved by the desirability function. Results: After optimization, the chromatographic separation was attained on Agilent Zorbax SB C18 (4.6×250mm, 5μm) column using gradient elution of mobile phase: (A) potassium dihydrogen phosphate (pH 2.7; 0.025M) and (B) acetonitrile at ambient column temperature with the last eluted compound in less than 17 min. The flow rate was maintained at 1/2.3 mLmin-1 with UV detection at 245/210 nm using time programming. Conclusion: The optimized chromatographic method was used for stability, indicating assay of the cited drugs in the presence of their related compounds according to ICH Q2R1 guidelines.