Current Chromatography - Current Issue

Aims: This study aimed to develop a high-performance liquid chromatography (HPLC) technique for estimating paracetamol glucuronide and paracetamol sulphate in the urine samples of preterm neonates. Background: Validated methods exist for estimating the principal metabolites of paracetamol in older children and those with liver disease. Here, we have developed and validated a simple technique for estimating the same in urine samples of preterm neonates. Objective: The study aims to develop and validate a simple, reliable, and accurate HPLC technique for estimating urinary paracetamol glucuronide and paracetamol sulphate metabolites. Methods: Preterm neonates of either sex diagnosed with patent ductus arteriosus (PDA) receiving paracetamol intravenously at the dose of 15 mg/kg every six hours were recruited. We ran the samples under standardized chromatographic conditions and using various dilutions of the calibration standards. Measures of assay selectivity, linearity, accuracy, and precision were estimated. Results: We observed that the peaks for paracetamol glucuronide and paracetamol sulphate were distinguished from those of the drug-free urine samples. The results for both metabolites revealed good reproducibility, with a percent coefficient of variation (% CV) of 4.3 and 4.9 for the slope for paracetamol glucuronide and paracetamol sulphate, respectively. Similarly, we observed good linearity, as indicated by the correlation coefficients of 0.99 for the metabolites. The validation assays revealed that the method is linear, accurate, and precise over the defined concentration ranges. Conclusion: We demonstrated that HPLC has good accuracy, reliability, and precision, and it can be used for estimating the principal metabolites from urine samples in neonates for defining the ontogeny of conjugation enzymes and in paracetamol overdose.

Background: The 6-mercaptopurine is an active ingredient used to treat certain types of leukemia. This drug is an immunosuppressive and antineoplastic agent that belongs to the thiopurine class. In Brazil, 6-MP is currently available only in the form of 50 mg tablets, sold as Purinethol® and manufactured by Glaxo Smith Kline. The lack of the liquid formulation’s production impedes treatment, assuming that one of its advantages is through its applicability in pediatric patients, who show the highest incidence among others. Objective: The purpose of this work was to evaluate the development and application of a reversed phase high performance liquid chromatography (HPLC) method using an Agilent 1220 Infinity® G4294B chromatograph with photodiode array detector. Methods: HPLC assays were performed on an Eclipse plus® C18 column (4.6 x 150 mm, 3.5 μm particle size) using a gradient mode mixture of acetonitrile and aqueous acetic acid solution as a mobile phase, with a flow of 1 mL.min-1 and detection at 324 nm. The method was validated by determining its selectivity, linearity, precision, accuracy, and robustness. Results: Retention time for 6-mercaptopurine was 5.12 minutes. The detector’s response was linear at concentrations from 1.6 to 2.4 μg/mL. The results of the method’s accuracy evaluation showed mean recovery of the amount of substance added to the samples of between 99.88 and 100.5%. For precision, repeatability and intermediate precision were evaluated. The repeatability showed a standard deviation of 0.0737. The intermediate precision was assessed on three different. For three days of the studies, the values of the standard deviations were less than 3%, showing repeatability and intermediate precision adequate for the analytical method in question. The limit of detection was determined as 3.6 ng/mL. The limit of quantification was determined as 12 ng/mL. The chromatographic method was robust. Conclusion: The proposed method can be applied to control the quality of 6-MP oral suspension to ensure that the required content is delivered to pediatric oncology patients.

Aims: This study aims to determine the quantitative prasugrel (PG) and its all possible process-related impurities. Background: To the best of our knowledge, very few analytical methods are available in the literature for monitoring process related impurities and degradation products of PG in bulk drug substance/ active pharmaceutical ingredient (API). Objective: The objective of this study is the separation of Prasugrel and its all possible process-related impurities viz., desacetyl prasugrel-tautomeric forms, intermediates including desacetyl impurity existing in its keto-enol form and positional tautomer impurities with degradation products. Methods: A simple and robust HPLC-UV method having Zorbax XDB C18 column (15 cm x 4.6 mm) 3.5μm particle size column was used. Results: Prasugrel and its process related impurities were separated as well as analyzed in pharmaceutical samples' biological matrices. Conclusion: RP-LC method was developed for quantitative determination of PG and related substantial impurities were found to be highly specific, sensitive and precise. The major oxidative degradant was identified as PG desacetyl IMPs (keto-enol and positional tautomer) and hydroxyl IMP.

Background: Levothyroxine is a synthetic thyroid hormone that is chemically identical to Thyroxine (T4), which is secreted by the follicular cells of the thyroid gland. Levothyroxine is used to treat deficiency of thyroid hormone and prevent the recurrence of thyroid cancer. Levothyroxine is present endogenously in the human body. Methods: It requires a treated matrix for the preparation of calibration curve standard and quality control samples. The method was developed using LC-MS/MS and validated in human charcoal stripped serum. Charcoal stripped matrix was used for the preparation of Calibration curve standards and Quality control samples. The method involves Solid-Phase Extraction technique. Levothyroxine D3 is used as an internal standard (ISTD). Results: Chromatographic separation was achieved using reversed phase analytical column Gemini NX-C18 110Å, 3μm (50x3.6) mm. Mobile phase consisted of acetonitrile and water in a ratio of 70:30 with 150μL of formic acid in 1000 mL of the mobile phase. Mobile phase achieved shorter run-time of 0.9 minute due to the use of Ultra-high performance liquid chromatography (UHPLC). Positive electro-spray ionization technique detected MRM ion pair transitions 777.60→731.65 for Levothyroxine and 780.70→734.6 for Levothyroxine- D3 (ISTD) were used. AB SCIEX Triple Quad™ API-4000 LC-MS/MS system and the bioanalytical method with 10ng/mL as the limit of quantification have been applied successfully to pharmacokinetics studies. Conclusion: Chromatographic separation was achieved using reversed phase analytical column Gemini NX-C18 110Å, 3μm (50x3.6) mm. Mobile phase consisted of acetonitrile and water in a ratio of 70:30 with 150μL of formic acid in 1000 mL of the mobile phase. Mobile phase achieved shorter run-time of 0.9 minutes due to the use of Ultra-high performance liquid chromatography (UHPLC). Positive electro-spray ionization technique detected MRM ion pair transitions 777.60→731.65 for Levothyroxine and 780.70→734.6 for Levothyroxine- D3 (ISTD). AB SCIEX Triple Quad™ API-4000 LC-MS/MS system and the bioanalytical method with 10ng/mL as the limit of quantification have been applied successfully to pharmacokinetics studies.

Aims: This study aimed to develop a new, fast and sustainable method by highperformance liquid chromatography (HPLC) for simultaneous determination of ascorbic acid and nicotinamide in the cosmetic emulsion. Background: Nicotinamide (NIC) and ascorbic acid (AA) are powerful antioxidants. AA presents excellent reducing power and protects the cell from oxidation. NIC is a precursor of NADPH and NADH, substances that present a strong reduction power, resulting in a huge antioxidant capacity. Objective: A new, fast and sustainable method using HPLC was developed and validated for simultaneous quantification of AA and NIC in the cosmetic emulsion. Methods: For this purpose, purified water with 0.01 % of trifluoracetic acid and ethanol (95:5, v/v), Symmetry Shield column (4.6 x 250 mm, 5 μm), 20 μL, 1.7 mL min^-1 at 254 nm were used. The method was validated according to the ICH, AOAC, and ANVISA, following parameters of linearity, detection and quantification limits, selectivity, accuracy, precision, and robustness. Results: The method was fast (2.7 min for AA and 3.2 min for NIC), linear between 20 and 80 μg mL^-1 (r = 0.9991 for AA and r = 0.9999 for NIC), precise (RSD < 5 % for AA and NIC), accurate (RSD 0.53 % for AA and 1.02 % for NIC), and selective for the emulsion base, and also robust to small changes in flow rate, injection volume, and purified water source. Conclusion: This work shows an ecologically alternative for the quantification of AA and NIC in the study of cosmetic emulsion by HPLC, which contemplates the requirements of the current green and sustainable analytical chemistry.

Background: Analysis of experimental retention data upon several variants of argentation liquid chromatographic separations of different mixtures of the same lipid class into their molecular species was made to estimate new parameters of their π-complexes for every unsaturated fatty acid residue as its silver ion cluster. Methods: Planar reversed-phase liquid chromatography, both in the presence and absence of silver ions as well as adsorption argentation liquid chromatography was applied for the separation of complex rac-1,2-diacylglycerol samples from three plant sources (cocoa butter, poppy seed, and linseed oils). Results: Every value of the argentation liquid chromatographic separation selectivity for any lipid molecular species upon both planar and column variants of reversed-phase fractionation of different complex samples from native sources into their molecular components is described by additive relative polarity levels of their fatty acid residues. These levels are always connected with equivalent lipophilicity values for every lipid molecule and its potential chemical variations during all variants of reversed-phase liquid chromatography in the presence of silver ion clusters. Conclusion: New parameters for several fatty acid residues of major native polyunsaturated lipid samples may be reflected by different coordination numbers of single silver atoms of its triangular pyramidal nanoclusters. Both hydrophobicity and total polarity levels of the coordination complexes of different lipid molecular species upon their adsorption argentation liquid chromatography may also be quantitatively estimated by their fixed methylene unit variations of these molecular species for two centigrade lipid scales.

Background: The combination of doxycycline (DOXY) and rifampicin (RIF) is recommended as a treatment therapy for brucellosis by the World Health Organization. Objective: The aim of the current study was to develop and validate the stability-indicating reversephase high-performance liquid chromatography (RP-HPLC) method for the analysis of a combination of doxycycline & rifampicin. Methods: The RP-HPLC method was developed and validated to estimate the doxycycline and rifampicin combination as per ICH guidelines. The drug combination solution was exposed to different stress conditions: acidic, basic, photo-oxidation, and oxidation. Results: The method was found linear in the range of 2 -10μg/mL for both the drugs with a retention time of 3.5 min for doxycycline and 6.5 min for rifampicin at lambda maximum of 350 nm. The RP-HPLC method was precise and accurate with %RSD < 2%. The intra-day and inter-day precisions were calculated and found within the acceptable range of 5%. Both drugs demonstrated good stability in the mobile phase after 6 hours. The LOD and LOQ of doxycycline and rifampicin were 100 ng/mL & 200ng/mL and 150ng/mL & 500ng/mL, respectively. The forced degradation of the combination of drug solutions was performed. The degraded drug peaks were well-resolved from the peaks of drugs. The percentage encapsulation efficiency of doxycycline and rifampicin in the nanoparticle system was assessed by utilizing the validated RP-HPLC method and found >60% (DOXY) and >70% (RIF). Conclusion: The developed RP-HPLC method of DOXY-RIF combination was rapid, accurate, precise, and stability-indicating. The method can be appropriately applied todetecte drugs in the nanoparticulate system.

The quality of drugs is a major concern for drug regulatory authorities and other stakeholders across the globe. Recently, drug regulatory authorities across the globe are facing a challenge in controlling the purity of cardiovascular (CVS) drugs for human use, especially drugs from the Angiotensin Receptor Blocker family, such as Valsartan. The present article aims to provide comprehensive knowledge on how pharmacopeias worldwide play a key role in ensuring the quality of active pharmaceutical ingredients (API) and finished pharmaceutical products (FPPs). In this article, the focus is on comprehensive information regarding pharmaceutical impurities, separation strategies, relevant regulatory guidelines to control impurities, and their acceptable limits, particularly with respect to cardiovascular active drug substances and drug formulations for human use.

Background: Abiraterone acetate is a derivative of steroidal progesterone, used as a firstline therapy for metastatic castration of prostate cancer. Objectives: The present study encompasses the design of an experiment approach for developing a simple, reliable, and rapid RP-HPLC method for the estimation of abiraterone acetate. Methods: The chromatographic separation was efficiently conducted on a Hypersil Gold C18 (50 x 4.6 mm, 5 μm) HPLC column, using the mobile phase composition of acetonitrile: dibasic potassium phosphate (0.01 mM) in the ratio of 80:20 (%v/v) at pH 6.5 with an isocratic elution mode. Furthermore, the different force degradation study including hydrolysis, oxidation, thermal, and photolytic was performed for abiraterone acetate. Results: The dynamic linearity was established in the concentration range of 0.5-10 μg/mL with r2 of 0.9998. Furthermore, the limit of detection and the limit of quantitation were 0.0978 μg/mL and 0.3260 μg/mL. The degradation of abiraterone acetate was shown in both acidic (54.16 ± 0.247 after 24 hrs) and basic conditions (35.06 ± 0.458 after 24 hrs). Furthermore, the developed method was successfully employed to quantify abiraterone acetate in bulk powder and the solid dispersion did not show any change in the retention time. Conclusion: The developed method was validated according to the ICH Q2 (R1) specification, which was found to be sensitive, accurate, precise, robust, linear, and selective compared to the reported chromatographic method.

Background: Kutki, the dried rhizome of Picrorhiza kurroa Royle ex Benth belonging to the family of Scrophulariaceae, has been utilized globally for liver ailments. Objective: Comprehensive use of kutki needs to evaluate its role as a quality control tool for discrimination of kutki samples, and therefore an effective HPLC fingerprinting method was established. Methods: Reverse-phase high-performance liquid chromatography with photodiode array (RPHPLC- PDA) detection method coupled with multivariate analysis was developed, which was modest, consistent and, accurate for classification of 11 kutki samples including authentic Picrorhiza kurroa rhizomes from the market of Ahmedabad and Gandhinagar in Gujarat, India. Results: The method was validated for various parameters like precision, reproducibility, and stability. The lowest value of the % relative standard deviations (RSD) was 1.31%. Chromatographic fingerprint profiles of 11 kutki samples, including authenticated samples, were obtained by this method, which showed a total of 28 peaks, and 9 peaks were important among them. Chemometric techniques like PCA and HCA were applied to identify the kutki samples.The Samples of kutki could be exquisitely differentiated into two clusters. Conclusion: HPLC-PDA method coupled with multivariate analysis divulged that chromatographic fingerprint analysis was reliable and effective for quality assessment and discrimination of kutki samples.