Sensitive UHPLC-MS/MS technique for monitoring Levothyroxine (T4) in human serum against endogenous thyroxin level

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 to prevent the recurrence of thyroid cancer. Levothyroxine is present endogenously in human body. Method: It requires 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. Method involves Solid-Phase Extraction technique. Levothyroxine D3 used as an internal standard (ISTD). Result: 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 mobile phase. Mobile phase achieved shorter run-time of 0.9 minute due to 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 limit of quantification has 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 mobile phase. Mobile phase achieved shorter run-time of 0.9 minute due to 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 limit of quantification has been applied successfully to pharmacokinetics studies.

Development of urinary assay methods for estimation of paracetamol glucuronide and paracetamol sulphate in preterm neonates with patent ductus arteriosus.

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.

Sulfated Polyborate, a novel buffer for low pH mobile phase on a non-end capped stationary phase in reverse phase liquid chromatography

Background: Sulfated Polyborate, a novel inorganic material primarily designed as a catalyst, has shown properties such as high solubility in organic solvents, low U.V. cut-off, and pKa ≈2.0, which suggests its potential as a mobile phase buffer for reverse-phase liquid chromatography. Objective: This study aims to substantiate the role of Sulfated Polyborate as mobile phase buffer for reverse-phase liquid chromatographic analysis of basic drugs with high pKa values viz. Bisoprolol fumarate, Timolol maleate, Verapamil hydrochloride, and Carvedilol. Methods: Solubilities, U.V. cut-offs, and pKa of Sulfated Polyborate was first experimentally confirmed. The behaviour of Sulfated Polyborate as mobile phase buffer at pH 3.0 was ascertained by varying the buffer concentration, flow rates, and percent organic modifier for elution of the four basic drugs on a non-end capped octyl silyl (C8) column. Similarly, the study was performed with KH2PO4 as a reference buffer. The column performance and conductometric measurements ascertained the impact of Sulfated Polyborate on the stationary phase. Results: Sulfated Polyborate and KH2PO4 buffers showed correlation coefficients of 0.99 and 1.00 for analyte retention factors for variation of buffer concentration and organic modifier composition, respectively. Peak symmetries and the number of theoretical plates were improved from > 2.0 to < 2.0 and ≈1000 to ≈3000, respectively, for Variation in buffer concentrations. Similar Van Deemter plots indicated equivalency of Sulfated Polyborate and KH2PO4 buffers. The column performance and conductometric measurements depicted no adsorption on the stationary phase. Conclusion: The present study demonstrates Sulfated Polyborate as a novel buffer for analytes with higher pKa on reverse-phase liquid chromatography.

Liquid Chromatographic analysis of Methotrexate and Minocycline- Relevance to the determination in Plasma/Nanoparticulate formulations

Aim: To develop an RP-HPLC method for the simultaneous estimation of methotrexate (MTX) and minocycline (MNC). Background: Different HPLC methods were reported for the estimation of MTX/MNC individually, but there is no report for the simultaneous estimation of both MTX and MNC in a simple method. Objective: The objective of the developed method is to utilize the method for the estimation of MTX/MNC in different pharmaceutical formulations and in biological fluids. Methods: An HPLC method for the estimation of methotrexate (MTX) and minocycline (MNC) relevance to the evaluation of nanoparticulate formulations has been developed and validated. Chromatographic estimation was achieved using the mobile phase composition of sodium acetate buffer and acetonitrile (70:30% v/v) at pH 4.0 at a flow rate of 0.2 mL/min at 307 nm. Results: The calibration curve for MTX and MNC was found to be linear at nanogram (5 to 25 ng.mL-1) and microgram (5 to 25 μg.mL-1) levels at a correlation coefficient range of 0.98 to 0.99 for both MTX/MNC. The lower limit of detection and limit of quantification were found to be 0.026 ng.mL-1 and 0.079 ng.mL-1 for MTX and MNC, respectively. The percentage relative standard deviation for validation parameters of both drugs was found to be less than 6.5%. The amount of MTX and MNC present within the nanoparticles was found to be MTX (0.84 mg/mL) and MNC (0.61 mg/mL). The in vitro release showed an immediate release pattern for MTX (64.95±2.08%) and MNC (90.90±1.78%) within 12 h. Conclusion: The developed analytical method for the simultaneous estimation of MTX and MNC was found to be simple, affordable, dynamic, low cost, rapid and easy to perform with good repeatability. This method is also time consuming, since the peaks were obtained within a moderate analysis time.

High Performance HPLC-UV Method Development and Validation for Sulfadoxine from its Potential Interfering Impurities

Objective: To address the separation of interfering potential impurities associated with the drug is always a daunting task. We present the method validation and quantitative determination of sulfadoxine (SUL), an anti-malarial drug with most important interfering impurities present pharmaceutical dosages and in bulk samples using HPLC-UV method. Methods: The UV detection was obtained at 270 nm and SUL is separated on Sunfire C18 (25 cm x 4.6 mm x 5 µ m) column at 45°C with flow rate of 1.0 mL/min in a mobile phase (CH3COOH:CH3CN). The stress testing (acidic/basic/oxidative) was performed using HPLC for SUL and its impurities showing the highly efficient separation peaks between degradant and drug product. Results: The developed method was found to be highly accurate and sensitive in regulation with ICH guidelines. Also, it was found to be free from interference from degradation products which allows the stability indicating capability of developed HPLC-UV method for SUL for validation in bulk drugs. Conclusion: The main advantages of the present method; (a) Separation achieved in 30 minutes, (b) MS compatible mobile phase renders this developed method can be directly adapted to LC-MS without any major modifications in near future, and (c) separation of twelve impurities on Sunfire C18 column. The CFs (correction factors) had been calculated for all the impurities. It was found to be 1.6 (IMP IX), 1.70 (IMP XI) and in between 0.8-1.3 for all other impurities. The LOD of the developed method for all the analytes were in the range of 0.05 to 0.11 μg/mL and the LOQ values were in the range of 0.17 to 0.36 μg/mL.

Development and Validation of a Simple Micromethod for the Determination of Metformin in Plasma by HPLC-UV for application in pharmacokinetics studies

Background: Metformin is the first-line drug to enhance glycemic control of type 2 diabetes mellitus (DM2) patients. Some reported methods to determine plasma metformin by HPLC-UV are not sensitive enough. Other methods require long extraction processes. Objective: The objective of this study was to develop and validate a simple and rapid analytical method to determine plasma metformin by HPLC-UV for application in a population pharmacokinetic study. Methods: Analyte was extracted from plasma by a simple protein precipitation technique using trichloroacetic acid (15%, w/v) as the precipitating agent. Plasma samples were analyzed using a C18 column (3.0 x 150 mm, 3.5 µm) under isocratic elution with 30 mM sodium hexansulfonate (pH 5) and acetonitrile (97: 3, v/v). Results: The limit of quantification (LOQ) was 0.1 µg mL-1 and the calibration curve was linear up to 4 µg mL-1 with a correlation coefficient >0.99. The mean recovery for metformin using this extraction procedure was 84.4 - 86.6%. The intra- and inter-day coefficients of variation and percent error values of the assayed method were <0% and <15% for LOQ and QCs, respectively. Metformin was stable in plasma samples by subjecting it to three freeze-thaw cycles and storing it up to 60 days at -80°C. This method was applied to determine plasma metformin concentrations in patients with type 2 diabetes mellitus treated with this drug. Conclusion: The HPLC-UV method developed is selective, accurate and precise for the quantification of metformin in plasma samples. Since sample, processing is fast and simple, in addition to being applicable in pharmacokinetic studies.

High-throughput, Determination of Vitamins (B1, B2, B3, B5, B6, B7 and B9) Using UPLC-ESI-MS.

Background: Multi-vitamin formulations are indicated for the elderly, adults, children’s and infants; to prevent or restore vitamin deficiencies. Objective: To provide a validated high-throughput method for simultaneous determination of vitamins in multi-vitamin formulations. Methods: A high-throughput UPLC-ESI-MS method was developed and validated for simultaneous determination of vitamins B1, B2, B3, B5, B6, B7 and B9. Analytes were eluted on an Acquity UPLC®BEH C18 1.7 μm, 2.1 x 50 mm column at 40 ± 5°C. Mobile phase containing acetonitrile (0.1% formic acid) and water (0.1% formic acid) in 30:70% ratio was pumped at 300 μL/min, under isocratic control. Protonated ions of vitamin B1, B2, B3, B5, B6, B7 and B9 were monitored in single ion recording mode, using electrospray ionization probe in positive mode. Results: The m/z ratio of positive ions of vitamin B1, B2, B3, B5, B6, B7 and B9 were 265.1, 377.2, 122.95, 220, 169.98, 244.9, and 442.1; respectively. The calibration curve of different linearity range (ng/ml) was prepared for each vitamin. Linearity range for vitamin B1, B2, B3, B5, B6, B7 and B9 were 60-1000, 25-1000, 75-5000, 30-1000 and 25-1000 ng/mL; respectively. Coefficient of variation for intra-day and inter-day precision for vitamin B1, B2, B3, B5, B6, B7 and B9; at middle and higher limit of quantitation were less than 15%. Conclusion: The method was successfully developed and validated, and three different brands of multi-vitamin tablets were assayed for water soluble vitamins.

Evaluation of the Efficiency of Protein A Affinity Chromatography to Purify a Monoclonal Antibody for Cancer Treatment and its Purity Analysis

Background:: Protein A affinity chromatography is often employed as the most crucial purification step for monoclonal antibodies to achieve high yield with purity and throughput requirements. Introduction:: Protein A, also known as Staphylococcal protein A (SPA) is found in the cell wall of the bacteria staphylococcus aureus. It is one of the first discovered immunoglobulin binding molecules and has been extensively studied since the past few decades. The efficiency of Protein A affinity chromatography to purify a recombinant monoclonal antibody in a cell culture sample has been evaluated, which removes 99.0% of feed stream impurities. Materials and Method:: We have systematically evaluated the purification performance by using a battery of analytical methods SDS-PAGE (non-reduced and reduced sample), Cation Exchange Chromatography (CEX), Size-exclusion chromatography (SEC), and Reversed phased-Reduced Chromatography for a CHO-derived monoclonal antibody. Results and Discussion:: The analytical test was conducted to determine the impurity parameter, Host Cell Contaminating Proteins (HCP). It was evaluated to be 0.015ng/ml after the purification step; while initially, it was found to be 24.431ng/ml. Conclusion:: The tests showed a distinct decrease in the level of different impurities after the chromatography step. It can be concluded that Protein A chromatography is an efficient step in the purification of monoclonal antibodies.

Distribution of Quercetin in Different Organs of Male Sprague Dawley Rats Under Hypoxia: A High-Performance Thin Layer Chromatography Approach

Background:: Quercetin (3’,3’,4,5,7-pentahydroxyflavonol), a natural flavonoid found in fruits, vegetables, beverages, and other phytoproducts, exerts multiple health benefits including a reduction in hypoxia-induced oxidative stress, inflammation, lipid peroxidation, allergic disorders, neurodegenerative disorders, and cardiovascular diseases. Objective:: Despite knowledge of such therapeutic efficacy of quercetin to human health, there is limited literature available that sheds light on an organ-wise distribution of quercetin. Therefore, the current study was performed to accurately estimate the distribution of quercetin in its supplemented form in different tissues of a mammalian model, i.e., male Sprague Dawley (SD) rats. Method:: The rats were exposed to different durations (1 h, 3 h, 6 h, and 12 h) of hypoxia in a simulated hypobaric hypoxia chamber, with parameters maintained at 8 % O2 and 282 mm Hg, following which they were sacrificed. Plasma and different tissue samples were duly collected. A high-performance thin layer chromatography (HPTLC) approach was employed for the first time, using our own reported method, along with an optimized sample preparation procedure for quercetin determination. Briefly, the samples were developed in a mobile phase constituted of ethyl acetate, dichloromethane, methanol, formic acid, and glacial acetic acid. Results:: Distinct bands of quercetin in resultant HPTLC profiles verified that the amount of quercetin varied among different tissues, with varying durations to hypoxia exposure. Quercetin was substantially retained in vital organs namely, lungs, liver, and heart for relatively longer durations. Conclusion:: The present study established HPTLC as an efficient and high throughput tool, leading to a satisfactory evaluation of the amount of quercetin present in various tissue samples under hypoxia.

Development and validation of an RP-HPLC method for the simultaneous determination of total withanolide glycosides and Withaferin A in Withania somnifera (Ashwagandha)

Background:: Withanolide glycosides in Ashwagandha (Withania somnifera), are important metabolites attributed with widely acclaimed therapeutic potential for which validated methods for quantitative determination are limited. Objective:: The primary objective was to develop and validate a Reversed-Phase High Performance Liquid Chromatography (RP-HPLC) method for simultaneous quantification of total withanolide glycosides (WG), withanoside IV and withaferin A present in ashwagandha extract.The study also aimed to identify various other constituents present in the extract. Materials and Methods: Aqueous methanol extract (AME) of Ashwagandha was prepared and fractionated into two viz. flavonoid rich fraction (FF) and withanolide rich fraction (WF). RP-HPLC method was developed and validated for the estimation of total WG in ashwagandha extract according to ICH guidelines. Preparative HPLC based purification of major compounds from WF fraction was carried out and constituents were identified using spectroscopic techniques. HPLC chemical profiling of WF before and after acid hydrolysis under controlled conditions was carried out to further confirm the glycosidic compounds. Results and Discussion: The RP-HPLC method gave a precise differentiation of flavonoids, withanolides and WG present in ashwagandha extract. The method demonstrated good reliability and sensitivity, and can be conveniently used for the quantification of total WG, withanoside IV and withaferin A present in ashwagandha extracts. According to this method, a purified fraction (WF) prepared from roots and leaves of Ashwagandha comprise 35% of total WG, 3.27% of withanoside IV and 2.40% of Withaferin A. The method was also applied to different products prepared from Ashwagandha with total withanolide glycosides ranged from 1.5% to 60%, and the results were found to be reproducible. Identification of the individual chemical constituents as well as the acid hydrolytic pattern of the extract further supported the reliability of the developed method for the quantitative determination of total WG. This study also reported a new withanolide glycoside named, cilistol V-6’-O-glucoside (Aswanoside) along with some other known withanolide glycosides. Conclusion:: A Reversed-Phase High Performance Liquid Chromatography (RP-HPLC) method was developed and validated for the quantitative determination of total WG, withanoside IV and withaferin A present in ashwagandha extract according to ICH guidelines. This study also reported a new withanolide glycoside named, cilistol V-6’-O-glucoside (Aswanoside) along with some other known WG.