Analytical Validation of an HPLC-UV Method for Praziquantel and Related Substances in PMMA-co-DEAEMA Microparticles

Validation of a Stability Indicating LC Method for Amiodarone HCL and Related Substances

A rapid and stability-indicating reversed phase high-performance liquid chromatography (RP-HPLC) method was developed for simultaneous quantification of paracetamol and ibuprofen in their combined dosage form especially to get some more advantages over other methods already developed for this combination. The method was validated according to United States Pharmacopeia (USP) guideline with respect to accuracy, precision, specificity, linearity, solution stability, robustness, sensitivity, and system suitability. Forced degradation study was validated according to International Conference on Harmonisation (ICH). For this, an isocratic condition of mobile phase comprising phosphate buffer (pH 6.8) and acetonitrile in a ratio of 65:35, v/v at a flow rate of 0.7 mL/minute over RP C18 (octadecylsilane (ODS), 150 × 4.6 mm, 5 μm, Phenomenex Inc.) column at ambient temperature was maintained. The method showed excellent linear response with correlation coefficient (R2) values of 0.999 and 1.0 for paracetamol and ibuprofen respectively, which were within the limit of correlation coefficient (R2 > 0.995). The percent recoveries for two drugs were found within the acceptance limit of (97.0–103.0%). Intra-and inter-day precision studies of the new method were less than the maximum allowable limit percentage of relative standard deviation (%RSD) ≤ 2.0. Forced degradation of the drug product was carried out as per the ICH guidelines with a view to establishing the stability-indicating property of this method and providing useful information about the degradation pathways, degradation products, and how the quality of a drug substance and drug product changes with time under the influence of various stressing conditions. The degradation of ibuprofen was within the limit (5–20%, according to the guideline of ICH), while paracetamol showed <20% degradation in oxidation and basic condition.

Mangiferin, a polyphenolic xanthone glycoside from Mangifera indica, is used as traditional medicine for the treatment of numerous diseases. The present study was aimed to develop and validate a reversed-phase high-performance liquid chromatography (RP-HPLC) method for the quantification of mangiferin from the bark extract of M. indica. RP-HPLC analysis was performed by isocratic elution with a low-pressure gradient using 0.1% formic acid: acetonitrile (87:13) as a mobile phase with a flow rate of 1.5 ml/min. The separation was done at 26°C using a Kinetex XB-C18 column as stationary phase and the detection wavelength at 256 nm. The proposed method was validated for linearity, precision, accuracy, limit of detection, limit of quantification, and robustness by the International Conference on Harmonisation guidelines. In linearity, the excellent correlation coefficient more than 0.999 indicated good fitting of the curve and also good linearity. The intra- and inter-day precision showed < 1% of relative standard deviation of peak area indicated high reliability and reproducibility of the method. The recovery values at three different levels (50%, 100%, and 150%) of spiked samples were found to be 100.47, 100.89, and 100.99, respectively, and low standard deviation value < 1% shows high accuracy of the method. In robustness, the results remain unaffected by small variation in the analytical parameters, which shows the robustness of the method. Liquid chromatography–mass spectrometry analysis confirmed the presence of mangiferin with M/Z value of 421. The assay developed by HPLC method is a simple, rapid, and reliable for the determination of mangiferin from M. indica.SUMMARYThe present study was intended to develop and validate an RP-HPLC method for the quantification of mangiferin from the bark extract of M. indica. The developed method was validated for linearity, precision, accuracy, limit of detection, limit of quantification and robustness by International Conference on Harmonization guidelines. This study proved that the developed assay by HPLC method is a simple, rapid and reliable for the quantification of the mangiferin from M. indica.Abbreviations Used:M. indica: Mangifera indica, RP-HPLC: Reversed-phase high-performance liquid chromatography, M/Z: Mass to charge ratio, ICH: International conference on harmonization, % RSD: Percentage of relative standard deviation, ppm: Parts per million, LOD: Limit of detection, LOQ: Limit of quantification.

During the survey of substandard medicines in Cambodia in 2007, it was found that more than 90% of 500-mg amoxicillin (AMPC) capsules failed the United States Pharmacopeia (USP) 30 TEST 1 dissolution test. In the USP, several monographs provide multiple methods for performing the dissolution test. By using the 500-mg AMPC capsule as an example, we aimed to identify the problems and implications of the USP methods adopted for the dissolution test as a global standard. All AMPC samples were collected from the Cambodian market in 2007. For the quantitative test, we referred to USP 30. We performed the USP 28 and USP 30 TEST 2 dissolution tests and compared these results with those of the USP 30 TEST 1. All 500-mg AMPC capsules used for the comparison passed the quantitative test. Samples that passed the USP 28 and USP 30 TEST 2 dissolution tests were identical, and the pass rate was 97.1% (34/35), whereas the pass rate with the USP 30 TEST 1 was 8.6% (3/35). The difference in the dissolution results between the three methods was significant (P<0.0001). This study revealed that many users would select the most stringent method when multiple methods exist in the USP. This may lead to a high failure rate of the tests. Because USP is a global standard, we recommend that it take into consideration the developing countries and create a more detailed user-friendly manual for selection for appropriate methods.

The main target of this current research work was intended to construct and make affirmation of a facile, economical, very sensitive with high precision and accuracy, reversed-phase high performance liquid chromatographic method for simultaneous estimation of two drugs Ramipril and Carvedilol. The validation parameters were verified based on the standard requirements of International Council for Harmonization (ICH), U.S. Food and Drug Administration (FDA) and United States Pharmacopoeia (USP) by the determination of linearity, accuracy and precision. To develop the method, a C18 column (with a dimension of 250 × 4.6 mm, 5 μ, SUFELCOSILTM LC-18) was used. The mobile phase was comprised of aqueous KH2PO4 buffer solution and acetonitrile at the ratio of 55:45 (V/V) and flow rate was 1 mL per minute. 220 nm wavelength in the ultra-violet region was used to monitor the effluents and the retention times were found at 5.1±0.1 and 8.1±0.1 minutes for Ramipril and Carvedilol, respectively. Percent recovery for both drugs was above 98%, which demonstrated that the accuracy protocol was maintained. The linearity responses of the method for both Ramipril and Carvedilol were higher than 0.995 and the percentage of Relative Standard Deviation (RSD) (precision) for both of these two drugs were lower than the highest permissible limit or less than 2% (according to FDA). Therefore, it is easily perceptible that the corresponding RP-HPLC method was highly accurate, effective, rapid and precise which can offer huge potential for the application of simultaneous assay of Ramipril and Carvedilol in pure forms. Bangladesh Pharmaceutical Journal 26(1): 15-19, 2023 (January)

This manuscript is about the identification of degradation products and four related substances of a novel antihistaminic drug Bilastine by LC-Q-TOF-MS/MS. A selective stability-indicating RP-HPLC method was developed for the separation of Bilastine, its 4 related substances and degradation products. Separation was done through Inertsil ODS-3® column (150 x 4.6 mm, 5 µm) using 10 mM ammonium acetate (pH 6.00) and methanol as eluent in a gradient at 1.0 mL/min flow rate. The chromatogram was extracted at 275 nm wavelength. Two degradation products and four related substances were identified and their structures were proposed on the basis of data obtained from LC-Q-TOF-MS/MS experiments. The developed method was validated in accordance with ICH Q2 and USP <1225> guidelines and it was found to be specific, precise, accurate and robust for the intended use. The method was linear in the range of 160-240 µg/mL for drug and 1.15-4.5 µg/mL for related substances with R2 value greater than 0.999 and 0.997 for drug and RS respectively. Percentage of relative standard deviation for validated parameters was less than 2. Structures of the drug, its degradation products and related substances were subjected to in silico studies for prediction of physicochemical, pharmacokinetic and toxicological properties with the help of QikProp module of Schrödinger suite 2017-1 software. Predicted in silico values suggested that they are safe and lie within the recommended range of parameters.

Five isocratic liquid chromatography (LC) methods have been examined for the separation of amoxicillin and its related substances on C18 or C8 columns. The United States Pharmacopeia (USP) assay method gave better selectivity. Similar selectivity was obtained not only on C18 columns but also on C8 and poly(styrene-divinylbenzene) columns. The good selectivity was confirmed by a second laboratory. A resolution test using cefadroxil was developed for the method performance. Based on the USP method, a gradient LC method was developed for the analysis of related substances in amoxicillin. This method has been proposed for the assay and purity control in the amoxicillin monographs of the European Pharmacopoeia and will be further examined in a collaborative study.

This is a pharmacokinetic study of Salviae miltiorrhizae and ligustrazine hydrochloride injection. The study aimed to evaluate the mechanism of action, safety and rational clinical use of Salviae miltiorrhizae and ligustrazine hydrochloride injection. Salviae miltiorrhizae and ligustrazine hydrochloride injection is a compound preparation consisted of Salvia miltiorrhiza extract and ligustrazine hydrochloride for the treatment of cardiovascular and cerebrovascular diseases in China. The study aimed to develop a rapid and sensitive high-performance liquid chromatography-diode array detector-tandem mass spectrometry (HPLC-DAD-MS/MS) method for simultaneous determination of six major active ingredients of Salviae miltiorrhizae and ligustrazine hydrochloride injection, namely danshensu, protocatechuic aldehyde, rosmarinic acid, lithospermic acid, salvianolic acid A, and ligustrazine hydrochloride, in rat plasma. Plasma samples were precipitated with methanol, which was spiked with ascorbic acid and the supernatant was separated on a Waters Cortecs C18 column, by using a gradient mobile phase system of acetonitrile-water containing 0.05% formic acid (v/v). For internal standards, puerarin was selected for the five salvianolic acids, while isofraxidin was used for ligustrazine hydrochloride. Besides, electrospray ionization in negative mode and multiplereaction monitoring were used to identify and quantify the five salvianolic acids, whereas ligustrazine hydrochloride was quantified at 310 nm using the diode array detector. Noticeably, all calibration curves showed good linearity (R2>0.99) over the concentration range, with a lower limit of quantification between 0.00411 and 0.0369 μg/mL for salvianolic acids, and 1.74 μg/mL for ligustrazine hydrochloride. Next, the precision of the developed method was evaluated by intra- and inter-day assays, and the percentage of relative standard deviation was within 10%. Although the extraction efficiency of some salvianolic acids was not very satisfactory, the sensitivity of the analytical method met the analysis requirements of rat plasma samples. Moreover, the validated method was successfully applied to a pharmacokinetic study of Salviae miltiorrhizae and ligustrazine hydrochloride injection in the rat model. Linear pharmacokinetic characteristics were observed for the six active ingredients after intravenous infusion administration in rats within the dose range examined here. In summary, our study proposed a HPLC-DADMS/ MS method with the simultaneous determination of multiple ingredients, and demonstrated its applicability in pharmacokinetic studies.

A simple yet sensitive high performance liquid chromatography method was developed and validated for the determination of paracetamol (PCM) content in different tablets dosage form. The mobile phase consisted of a mixture of methanol: water (80:20 v/v). The HPLC analysis was performed at a flow rate of 1 mL/min using a Thermo Synchronise C18 (150 × 4.6mm, 5μM) and an UV detection wavelength of 254 nm was used. The method was validated for selectivity, linearity, precision, accuracy, limit of quantification (LOQ), limit of detection (LOD), robustness and solution stability. The calibration curve was linear over a concentration range of 20 to 70 µg/mL (r2 = 0.9997) with LOD and LOQ of 0.01 and 0.03 μg/mL, respectively. The retention time for paracetamol was 1.5 min. The relative standard deviation percent (%RSD) and the relative error percent (%RE) of calibration curve were between 0.03-1.03% and 0.05-1.11%, respectively. The intra- and inter-day precision and accuracy were between 0.02-1.55% and 0.10-0.78%, respectively. In addition, the %RSD of short term stability and the method robustness were between 0.4-1.20% and 0.09-1.43%, respectively. All results followed the ICH guideline Q2 (R1) which indicates the precision and accuracy of the developed method. The method was successfully applied to assess PCM content in different marketed tablets in Iraq.

<b>Perspectives on Method Validation</b>: Validation is a Multistep Process with USP Regulatory Guidelines at each Step

An accurate, precise, sensitive and reproducible High-performance liquid chromatographic (HPLC) and UV spectrophotometric methods were developed and validated for the quantitative determination of haloperidol (HPD) in bulk drug and pharmaceutical formulation. Different analytical performance parameters such as linearity, precision, accuracy, specificity, limit of detection (LOD) and limit of quantification (LOQ) were determined according to International Conference on Harmonization ICH Q2B guidelines. The RP-HPLC method was developed by the isocratic technique on a reversed-phase Thermo C18 (250 × 4.6 mm, 5µm) column with mobile phase consisting of Methanol: Acetonitrile (50:50v/v) at flow rate of 1.0 ml/min. The retention time for HPD was 2.238±0.3min. The UV spectrophotometric determinations were performed at 244 nm using 80% methanol as a solvent. The linearity range for HPD was 5-25 μg/ml for both HPLC and UV method. The linearity of the calibration curves for each analyte in the desired concentration range was good (r2 &gt;0.999) by both the HPLC and UV methods. The method showed good reproducibility and recovery with percent relative standard deviation less than 2%. Moreover, the accuracy and precision obtained with HPLC co-related well with the UV method which implied that UV spectroscopy can be a cheap, reliable and less time consuming alternative for chromatographic analysis. The proposed methods are highly sensitive, precise and accurate and hence successfully applied for determining the assay and in vitro dissolution of a marketed formulation.&#x0D; Keywords: HPLC, UV Spectrophotometry, Haloperidol, Pharmaceutical formulation, Method validation, Quantitative analysis

The stability-indicating method for Deflazacort Suspension was developed using the Analytical Quality by Design (AQbD) approach, focusing on process control and understanding. A multilevel factorial design was used to optimize the gradient mode's time and mobile phase ratio. Sophisticated software like Design Expert and Minitab aided in chromatographic condition optimization. The method utilizes a Zorbax Eclipse XDB C18 column (150mm x 4.6mm, 5µm particle size), with a flow rate of 1.0 mL/min, and monitoring the analyte with a UV/PDA detector at of 245nm wavelength known for its reliability. It employs two mobile phases: A, consisting of Water, Tetrahydrofuran, and Acetonitrile (91:3:6, v/v/v), and B, comprising Water, Tetrahydrofuran, Acetonitrile, and Methanol (4:2:74:20, v/v/v/v). Statistical analysis confirms the importance of these components in achieving effective separation and detecting related substances. The validation of method was done as per ICH guidelines. Linearity, specificity, Limit of Detection (LOD), Limit of Quantification (LOQ), precision, accuracy, solution stability, and robustness parameters were validated. The method was validated to ensure reliability and accuracy, demonstrating linearity from 0.02 to 1.2ppm with a correlation coefficient (r2 &gt; 0.99) at 245nm. This confirms the method's ability to precisely quantify related substances in Deflazacort Suspension. The stability of Deflazacort Oral Suspension was rigorously assessed, including a forced degradation study. Critical system suitability parameters, tailing factor, and theoretical plate, met acceptable limits, confirming the method's efficiency and resolution. Successful separation of degradation peaks from each other and the main peak was achieved during the study. The method's robustness was confirmed with variations under 2%, ensuring consistent and reproducible results despite minor changes in conditions. Peak purity analysis showed no co-eluting peaks, confirming the method's specificity. These findings validate the stability-indicating nature of the chromatographic method for Deflazacort Oral Suspension analysis, ensuring its reliability for quality control.

Objective: The present paper describes a simple, accurate, and precise reversed-phase high-performance liquid chromatography (HPLC) method for rapid and simultaneous quantification of netupitant (NTP) and palonosetron (PLS) in bulk and pharmaceutical dosage form.&#x0D; Methods: The chromatographic separation was achieved on Luna C18 (250 mm × 4.6 mm, 5 μ). Mobile phase contained a mixture of 0.1% orthophosphoric acid and acetonitrile in the ratio of 60:40 v/v, flow rate 1.0 ml/min, and ultraviolet detection at 222 nm.&#x0D; Results: The proposed method shows a good linearity in the concentration range of 60–900 μg/ml for NTP and 0.1–1.5 μg/ml for PLS under optimized conditions. All the precision and recovery results are in between 98 and 102%. In the entire robustness conditions, percentage of relative standard deviation is &lt;2.0%. Degradation has minimum effect in stress condition and solutions are stable up to 24 h. This method is validated different parameters such as precision, linearity, accuracy, limit of detection, limit of quantification, ruggedness, robustness, and forced degradation study were determined according to the International Conference of Harmonization (ICH) Q2B guidelines.&#x0D; Conclusion: All the parameters of validation were found to be within the acceptance range of ICH guidelines. Since there is no HPLC method reported in the literature for the estimation of NTP and PLS in pharmaceutical dosage forms, there is a need to develop quantitative methods under different conditions to achieve improvement in sensitivity, selectivity, etc. Hence, the author has attempted to develop a validation and forced degradation for simultaneous quantification of NTP and PLS.

The high worldwide consumption of antibiotics and their complex impurity profiles has drawn the attention of the scientific community to the development and validation of stability methods for these drugs. Amphotericin B is an antibiotic that is a natural fermentation product of bacterium Streptomyces nodosus, used as a broad-spectrum antifungal agent, and is highly unstable. For this reason, the objective of this work is the development and validation of an indicative method of stability by high performance liquid chromatography (HPLC) associated to diode array detector (DAD) for amphotericin B (AMB). To achieve this, the chromatographic profiles of acid, basic, oxidative and thermal degradation caused by AMB exposure to water and light were verified by HPLC-DAD, using an isocratic method under the following conditions: C18 chromatographic column (200 × 4.6 mm-5 µm), mobile phase composed of 65:35 of organic phase (methanol/acetonitrile in 41:18)/aqueous phase (2.5 mmol L-1 of disodium edetate, pH 5.0), flow rate of 1.0 mL min-1, injection volume of 20 µL, column temperature 30 ± 2 °C and wavelength of 383 nm. After identification of these profiles, the method was validated according to recommendations of the International Conference on Harmonization guidelines, and then, the active pharmaceutical ingredient (API) peak purity grade, percentage of drug degradation, occurrence of impurities peak and its identification by mass spectrometry (MS) with electrospray ionization (ESI), under positive ionization mode, were evaluated. It is suggested that the main degradation products were amphotericin B (2), degradation product 1 (DP1) and degradation product 2 (DP2) in acid and oxidative medium. Amphotericin B was stable in the presence of water and at 70 ± 2 °C for seven days.

The aim of the study was to develop a method for fast and reliable diagnosis of peroxisomal diseases and to facilitate differential diagnosis of cholestatic hepatopathy. For the quantification of bile acids and their conjugates as well as C(27) precursors di- and trihydroxycholestanoic acid (DHCA, THCA), in small pediatric blood samples we combined HPLC separation on a reverse-phase C18 column with ESI-MS/MS analysis in the negative ion mode. Analysis was done with good precision (CV 3,7%-11.1%) and sufficient sensitivity (LOQ: 11-91 nmol/L) without derivatization. Complete analysis of 17 free and conjugated bile acids from dried blood spots and 10 microL serum samples, respectively, was performed within 12 min. Measurement of conjugated primary bile acids plus DHCA and THCA as well as ursodeoxycholic acid was done in 4.5 min. In blood spots of healthy newborns, conjugated primary bile acids were found in the range of 0.01 to 2.01 micromol/L. Concentrations of C(27) precursors were below the detection limit in normal controls. DHCA and THCA were specifically elevated in cases of peroxysomal defects and one Zellweger patient.