Simultaneous Quantitative Determination of Five Process Relevant Impurities in Menatetrenone

Literature survey reveals that few spectrophotometric, high performance liquid chromatographic (HPLC)and High performance thin layer chromatographic (HPTLC)methods were reported for the estimation of Amlodipine and perindopril in bulk and combined pharmaceutical dosage forms. But there is no any stability indicating high performance liquid chromatography study is reported for amlodipine. So the present study was aimed to development of simple, accurate, rapid and economical HPLC stability indicating assay method were established for the determination of Amlodipine and Peridopril in bulk and tablet dosage form. A High-performance liquid chromatograph WATERS, software: Empower 2,2695 separation module, 996 PDA detector, using Phenomenex Luna C18 (4.6mm×250mm) 5µm or equivalent column, with mobile phase composition of Methanol: Phosphate Buffer pH3.0 (70:30v/v) was used. The flow rate of 1.0ml min-1 and effluent was detected at 230nm. The retention time of Amlodipine and Perindopril was found to be 1.870min and 2.499minutes respectively. Linearity was observed over concentration range of 10-50µg ml-1 for Amlodipine and 16-80µg ml-1 for Perindopril respectively. The accuracy of the proposed method was determined by recovery studies and the Amlodipine was found to be 99.1% and Perindopril was found to be 98.8% respectively. The proposed method is applicable to routine analysis of Amlodipine and Perindopril in bulk and pharmaceutical formulations. The proposed method was validated for various ICH parameters like linearity, limit of detection, limits of quantification, accuracy, precision, range and specificity.

The purpose of this study is to develop and validate a method based on size‐exclusion chromatography (SEC) for the simultaneous determination of fatty acid methyl esters (FAME), monoacylglycerols (MAG), diacylglycerols (DAG), and triacylglycerols (TAG) in biodiesel. The proposed method presents good linearity. The limits of detection are 0.26% mass for FAME, 0.02% mass for MAG, 0.01% mass for DAG, and 0.02% mass for TAG. The limits of quantification are 0.78% mass for FAME, 0.06% mass for MAG, 0.01% mass for DAG, and 0.06% mass for TAG. Accuracy evaluated by recovery yielded values ranging from 98.93% to 117.67%. Precision is evaluated by repeatability (%), which is ranged from 0.03% to 13.67%. The proposed SEC method proves effective in determining the FAME, MAG, DAG, and TAG content of standard samples, and the paired t‐test shows that the results obtained were statistically similar to the gas chromatography (GC) values. The method also has some advantages over the reference GC methods, since it obtains the content for each class analyzed, irrespective of its components. Also, it does not require derivatization, which makes it easier and also quicker (15 min) than the 60 min taken by the two reference methods, and it does not need an internal standard, which makes it cheaper.Practical Applications: Size exclusion chromatography (SEC) is an efficient method for simultaneous and quantitative determination of fatty acid methyl esters (FAME), monoacylglycerols (MAG), diacylglycerols and triacylglycerols (TAG). The method present itself as an alternative to reference methods (ASTM D 6584 and ABNT NBR 15764) based on gas chromatography (GC). The proposed method shows advantages compared to reference methods, once it makes possible to determine the content of each constituent class in samples, regardless of its components, what makes the peak integration easier. Beyond that, previous sample derivatization is unnecessary, what makes the method simpler, cheaper and faster (15 min) than both reference methods that demands together 60 min for analysis (ASTM D 6584 for MAG, DAGa and TAG and ABNT NBR 15764 for FAME analysis).

Atopic dermatitis is a typical chronic inflammatory skin disease that affects all age groups and requires basic skin care for treatment. Anti-inflammatory and antiallergy steroids are the most frequently used treatments but they are limited due to their side effects caused by a weakening of the immune system. Many consumers focus on performance as a criterion for selecting cosmetics. However, steroids have been illegally used to improve the performance of cosmetics, and consumers have been adversely affected by the corresponding side effects. In this paper, we propose a simple and rapid method using liquid chromatography-tandem mass spectrometry to simultaneously analyze ten non-permitted atopic therapeutic compounds in cosmetic products: chlorpheniramine maleate, ketotifen fumarate, doxepin hydrochloride, azelastine hydrochloride, bufexamac, clotrimazole, tranilast, fusidic acid, tacrolimus, and pimecrolimus. Additionally, the major characteristic fragment ions for tacrolimus, pimecrolimus, and clotrimazole were identified by time-of-flight mass spectrometry. The specificity, linearity, limit of detection, limit of quantification, recovery, precision, accuracy, and stability of the proposed method were validated. The limit of detection and quantification were in the ranges of 5.05-203.30 pg/mL and 15.15-609.90 pg/mL, respectively. The proposed analysis method could help improve the safety management of cosmetics.

Determination of optimal sample preparation for aldehyde extraction from pale malts and their quantification via headspace solid-phase microextraction followed by gas chromatography and mass spectrometry

A multiresidue method based on extraction with organic solvents, cleanup by preparative liquid chromatography, and detection by gas chromatography (GC) using tandem mass spectrometry (MS/MS) mode is described for the determination of α- and β-endosulfan and three main metabolites (sulfate, ether, and lactone) in human adipose tissue samples. The analytical methodology is verified, and the values of some performance characteristics, such as linearity, limit of detection (LOD), limit of quantification (LOQ) limits, precision (intraday and interday), and accuracy (recovery) are calculated. The high efficiency of the cleanup step for the elimination of interference allows reaching detection limits at low micrograms per kilogram (parts per billion, ppb) concentration levels. In addition, an estimation of measurement uncertainty, using validation data, is presented for each target compound. The results show that the sources of largest uncertainty are those relative to the balance calibration, from the gravimetric step, and both the relative uncertainty associated with the recovery and the intermediate precision of the method.

ABSTRACTIntroduction Acetonitrile (ACN) and ethanol (EtOH) are common solvents used in radiopharmaceutical production. In accordance to official compendia, the concentration of these solvents should be assessed by gas chromatography. In the present paper, an optimized method, based on Koziorowski (2010), is validated. Methods ACN and EtOH concentrations and retention times (Rt) were obtained by a HPLC system equipped with a refractive index detector (RID), an ion exclusion column and ultrapure water as mobile phase. The methodology was validated following the ICH Q2(R1) requirements. Results The solvents EtOH and ACN were eluted at 23.22 and 26.32 minutes, respectively, with a final run time of 30 minutes. The validation parameters (accuracy, precision, linearity, specificity, robustness, detection and quantification limits) were obtained. Conclusions A reproducible HPLC method for the quantification of residual solvents in preparations of 2-deoxy-2-[fluorine-18]fluoro-D-glucose (18F-FDG) was described and validated. The method was precise, accurate, selective, robust and linear over a wide range. In addition, this method showed a high sensitivity, with limits of detection and quantitation comparable to the usual methods by gas chromatography.

The primary objective of the present investigation is to develop and validate a simple, robust, and cost-effective isocratic reverse phase-high-performance liquid chromatography (RP-HPLC) method for determining isoliquiritigenin (ISL) in both bulk and biological samples using an analytical quality-by-design (AQbD) approach. The central composite design was employed for method optimization using Design Expert® software, by taking mobile phase ratio and flow rate as independent variables and peak area, retention time, tailing factor, and theoretical plates as dependent variables. The design suggested the use of a mobile phase consisting of acetonitrile:0.2% ortho-phosphoric acid (75:25, v/v) and a flow rate of 0.9 mL/min as optimal chromatographic conditions. The detection of ISL was performed at 364 nm. The optimized method was validated in accordance with International Conference on Harmonization (ICH) Q2(R1) guidelines. The method showed excellent linearity, limit of detection, limit of quantification, accuracy, precision, robustness, and system suitability. All validation parameters fell within the acceptable limits set by ICH. Additionally, the applicability of the method in biological samples were analyzed. In conclusion, the results suggest that the developed and validated AQbD-based RP-HPLC method was well-suited for the estimation of ISL in bulk and biological sample.

Background:Dianthus superbus, one of traditional herbal medicine, is widely used to treat urethritis, carbuncles and carcinoma.Objective:A simultaneous determination method was established for controlling the quality of D. superbus using the eight compounds, (E)-methyl-4-hydroxy-4-(8a-methyl-3-oxodecahydronaphthalen-4a-yl) (1), diosmetin-7-O(2'',6''-di-O-α-L-rhamnopyranosyl)-β-D-glucopyranoside (2), vanillic acid (3), 4-hydroxyphenyl acetic acid (4), 4-methoxyphenyl acetic acid (5), (E)-4-methoxycinnamic acid (6), 3-methoxy-4-hydroxyphenylethanol (7), and methyl hydroferulate (8) isolated from D. superbus.Materials and Methods:This analysis method was developed using high performance liquid chromatography coupled with diode array detector with a Shishedo C18 column at a column temperature of 3°C. The mobile phase was composed of 0.1% trifluoroacetic acid in water and acetonitrile. The flow rate was 1 ml/min and detection wavelength was set at 205 nm and 280 nm. Validation was performed in order to demonstrate selectivity, accuracy and precision of the method.Results:The calibration curves showed good linearity (R2 > 0.99). The limits of detection and limits of quantification were within the ranges 0.0159–0.6205 μg/ml and 0.3210–1.8802 μg/ml, respectively. Moreover, the relative standard deviations of intra- and inter-day precision were both <2.98%. The overall recoveries were in the range of 96.23–109.87%. Quantitative analysis of eight compounds in 12 D. superbus samples (D-1–D-12) from various regions were analyzed and compared by developed method.Conclusion:As a result, this established method was accurate and sensitive for the quality evaluation of eight compounds isolated from D. superbus and may provide a new basis for quality control of D. superbus.SUMMARYA simultaneous determination method of eight compounds in Dianthus superbus was established by high performance liquid chromatography-diode array detectorDeveloped analysis method is validated with linearity, precious and accuracyThe newly established method was successfully evaluated contents of eight compounds in 12 D. superbus samples (D.1.D.12) from various regions and compared.Abbreviations used: HPLC: High performance liquid chromatography, LOD: Limits of detection, LOQ: Limits of quantification, RSD: Relative standard deviation.

Accurate quantification of free fatty acids in dairy products is important for both product quality control and legislative purposes. In this study, a novel fatty acid butyl ester method was developed, where extracted free fatty acids are converted to butyl esters prior to gas chromatography with flame ionization detection. The method was comprehensively validated to establish linearity (20-700 mg/L; R2 > 0.9964), limits of detection (5-8 mg/L), limits of quantification (15-20 mg/L), accuracy (1.6-5.4% relative error), interday precision (4.4-5.3% relative standard deviation), and intraday precision (0.9-5.6% relative standard deviation) for each individual free fatty acid. A total of 17 dairy samples were analyzed, covering diverse sample matrices, fat content, and degrees of lipolysis. The method was compared to direct on-column injection and fatty acid methyl ester methods and overcomes limitations associated with these methods, such as either column-phase absorption or deterioration, accurate quantification of short-chain free fatty acids, and underestimation of polyunsaturated free fatty acid.

Background Treosulfan and busulfan are alkylating agents administered as the conditioning regimen before hematopoietic stem cell transplantation. High busulfan exposure is associated with drug toxicity and low busulfan exposure with rejection or disease recurrence. There is also an association between treosulfan exposure and early toxicity, such as skin toxicity and mucositis. As both busulfan and treosulfan have narrow therapeutic ranges, therapeutic drug monitoring (TDM) is essential. Since the clinical laboratory tests for both drugs are few, a method that can simultaneously measure both drug concentrations under the same conditions would be helpful in practice. In this study, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous measurement of treosulfan and busulfan was developed and validated. Methods For preparing calibrators and quality control materials, commercialized reference standards were used for treosulfan, an active metabolite of treosulfan, S,S-EBDM, and busulfan. Treosulfan-D4 and busulfan-D8 isotopes were used as internal standards (ISs). The analytes were separated from 25 µL of plasma after protein precipitation with acetonitrile containing ISs. After centrifugation, the supernatant was diluted with 3% formic acid and then injected into the liquid chromatography system followed by tandem mass spectrometry. Chromatographic separation was performed using ACQUITY Ι-Class plus ultraperformance liquid chromatography (UPLC) system coupled with a XEVO TQ-XS mass spectrometer operated in positive ion electrospray ionization mode. The analytes and ISs were detected in the multiple reaction monitoring (MRM) mode. The limit of detection (LoD), limit of quantification (LoQ), selectivity, linearity, accuracy, precision, carryover, matrix effect, and sample stability were validated according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. Results Retention times for treosulfan, S,S-EBDM and IS for both analytes were 0.44 min, 0.43 min, and 0.44 min, respectively. Retention times for busulfan and IS for busulfan were 1.20 min and 1.18 min, respectively. The mass-to-charge (m/z) transitions used for quantification in the MRM mode were as follows: treosulfan 296.2 &amp;gt; 87.1; S,S-EBDM 200.2 &amp;gt; 87.1; and busulfan 264.1 &amp;gt; 55.1. The total run time was 4 min. The LoDs of treosulfan, S,S-EBDM, and busulfan were 0.21, 0.23, and 0.001 µg/mL, respectively. The linear range of the calibration curves of treosulfan, S,S-EBDM, and busulfan spanned concentrations of 3.13–100, 0.63–5.00, and 0.16–5.00 µg/mL, respectively. The total precision of the developed method fulfilled the analytical criteria, except for S,S-EBDM. The LC-MS/MS method was adequately selective and accurate and showed no carry-over. It provided an acceptable matrix effect for treosulfan and busulfan; however, the matrix effect could not be excluded for S,S-EBDM. Busulfan and treosulfan were stable in plasma for 2 h at room temperature. When the samples were stored at 10 °C, busulfan was stable for up to a week and treosulfan was stable for up to 3 days, and both were stable for up to a week when stored frozen at −20 °C. Conclusion We developed the LC-MS/MS method to simultaneously measure treosulfan and busulfan. The method met the validation requirements of the CLSI guidelines and showed good performance. Therefore, this method is expected to be helpful in TDM after treosulfan or busulfan treatment in clinical laboratories.

A sensitive, specific, and rapid high-performance liquid chromatography (HPLC) method for the determination of ambrisentan enantiomers has been developed and validated. Six chiral columns were tested in a reversed-phase system. Excellent enantioseparation with the resolution more than 2.5 was achieved on Chiralcel OZ-3R (cellulose 3-chloro-4-methylphenylcarbamate) using mixture of 20 mM sodium formate (pH 3.0) with acetonitrile (55:45; v/v). Validation of the HPLC method including linearity, limit of detection, limit of quantification, precision, accuracy, and selectivity was performed according to the International Conference on Harmonisation (ICH) guidelines. The method has an advantage of a very quick chromatographic separation (less than 6 min) and therefore is highly suitable for routine determination of (R)-ambrisentan in enantiopure active pharmaceutical ingredient (S)-ambrisentan.

This investigation is aimed to develop and validate a high-performance thin layer chromatography method for quantitative determination of serum zonisamide levels. Chromatographic separation was carried out on a silica gel 60F254 plate using a mixture of ethyl acetate:methanol:toluene (4:1:5) as the mobile phase. Densitometric detection was carried out at 254 nm. The method was validated for linearity, precision, selectivity, limit of detection, limit of quantification and accuracy. Linear calibration curves in the range of 5 to 80 µg/band gave a correlation coefficient of 0.991. The intra-day (n=6) and inter-day (n=18) precision, expressed as the relative standard deviation were in the range of 2.83 to 3.27 % and from 2.09 to 4.39 %. The limits of detection and quantification were found to be 1.07 and 3.15 µg/band, respectively. Accuracy was calculated as percent recovery and was found to be 97.52 and 115.23 %. Theophylline was used as an internal standard, which gave a well separated peak at Rf 0.36 without interfering with zonisamide. The method was found to be specific with no matrix interference. Thus, the method developed for the estimation of serum zonisamide level is simple, cost-effective and reliable for therapeutic drug monitoring.

Validation of a headspace solid-phase microextraction procedure with gas chromatography-electron capture detection of pesticide residues in fruits and vegetables

Objective: Nitrosamine impurities, particularly nitrosamine drug substance-related impurities (NDSRIs), have gained focus in recent past due to their carcinogenic potential. Due to the low sensitivity of conventional techniques like high-performance liquid chromatography (HPLC) and gas chromatography (GC), the exact levels of these impurities could not be determined. This study aims to develop and validate a sensitive liquid chromatography hyphenated with mass spectrometry (LC-MS/MS) method to estimate the trace levels of N-Nitroso fluoxetine (a fluoxetine-related NDSRI) in fluoxetine capsules (60 mg). Methods: We developed a sensitive and selective LC-MS/MS method for the quantification of trace levels of N-Nitroso fluoxetine in fluoxetine capsules (60 mg) using the AB Sciex 5500+quadrupole ion trap (QTRAP) LC-MS/MS system. To achieve the maximum response at quantitation level for N-Nitroso fluoxetine impurity, chromatographic conditions optimized by selecting a specific combination of column, mobile phase, and diluent. Mobile Phase A and B consisted of 10 mmol ammonium acetate buffer and methanol, respectively. Waters X-Bridge C18 column (250 × 4.6 mm, 3.5 μm, Part No. 186003943) at temperature 40 °C and mobile phase flow rate to 0.5 ml/min has yielded consistent results by employing an optimized gradient elution program (Tmin/%B): 0/75, 2/75, 16/95, 28/98, 28.1/75, and 35/75 for the analysis. To further maximize the response of the N-Nitroso fluoxetine impurity, the multiple ion reaction monitoring (MRM) mass spectrometry method in positive electron spray ionization (ESI) mode is preferred over single ion monitoring (SIM) method. Results: The newly developed method is validated by performing parameters such as specificity, method precision, intermediate precision, detection limit, quantitation limit, linearity, and accuracy in accordance with the international conference on harmonization (ICH Q2 (R1)) Guidelines. Limit of detection (LOD) and limit of quantitation (LOQ) are established as 0.06 ppm and 0.11 ppm, respectively. The method is found capable to demonstrate accuracy, precision, and linearity across the range of 0.11 to 1.68 ppm (10% to 150% of the specification), where linearity study shows the correlation coefficient (r²) greater than 0.99 for linearity plot between concentration and area. Recovery data at 10%, 50%, 100%, and 150% of the specification levels indicates that the recovered impurity content observed between 80% and 120%. Based on these results, we have successfully established the method for batch analysis of fluoxetine capsules (60 mg). Conclusion: A new rapid and sensitive reverse-phase ultra-high-performance liquid chromatographic (UHPLC) method coupled with a mass spectrometry detector has been developed and validated for the estimation of N-Nitroso fluoxetine impurity in fluoxetine capsules. Method validation is accomplished according to the ICH Q2 (R1) guidelines, assessing parameters such as limit of detection, limit of quantitation, accuracy, precision, and linearity. We found the method suitable for its intended use and successfully applied it to test batches of the fluoxetine capsules drug product.

Objectives: In this work, a new, rapid, simple, precise and specific method has been developed for the simultaneous determination of amlodipine (AML) and rosuvastatin (ROS) in pharmaceutical preparations by square wave voltammetry (SWV). Methods: Electrochemical behavior and simultaneous voltammetric determination of AML and ROS were investigated using platinum disk electrode. Validation parameters such as specificity, linearity, accuracy, precision, ruggedness, stability, limit of quantification and limit of detection were studied according to the ICH Guidelines. Results: The linearity of this developed method was established in the concentration range of 5-40 μg/mL for AML and ROS, respectively. The precision was less than 1.88 and 1.93%, determined from quality control samples for AML and ROS, and accuracy was within 1.69 and 1.97% in terms of relative error, respectively. The percentage recovery obtained for AML and ROS in pharmaceutical preparations were 99.5 and 100.2%, respectively. Limit of detection and quantification for AML were 0.70 and 2.10 µg/mL, for ROS 0.80 and 2.40 µg/mL, respectively. Conclusion: The developed SWV method can be used for routine analysis of AML and ROS in pharmaceutical preparations.