The Development and Validation of a Stability-Indicating UHPLC-DAD Method for Determination of Perindopril l-Arginine in Bulk Substance and Pharmaceutical Dosage Form

A stability-indicating ultra-high-performance liquid chromatography (UHPLC) method with a diode array detector was developed and validated for the determination of cis/trans isomers of perindopril l-arginine in bulk substance and pharmaceutical dosage form. The separation was achieved on a Poroshell 120 Hilic (4.6 × 150 mm, 2.7 µm) column using a mobile phase composed of acetonitrile–0.1 % formic acid (20:80 v/v) at a flow rate of 1 mL min⁻¹. The injection volume was 5.0 µL and the wavelength of detection was controlled at 230 nm. The selectivity of the UHPLC-DAD method was confirmed by determining perindopril l-arginine in the presence of degradation products formed during acid–base hydrolysis and oxidation as well as degradation in the solid state, at an increased relative air humidity and in dry air. The method’s linearity was investigated in the ranges 0.40–1.40 µg mL⁻¹ for isomer I and 0.40–2.40 µg mL⁻¹ for isomer II of perindopril l-arginine. The UHPLC-DAD method met the precision and accuracy criteria for the determination of the isomers of perindopril l-arginine. The limits of detection and quantitation were 0.1503 and 0.4555 µg mL⁻¹ for isomer I and 0.0356 and 0.1078 µg mL⁻¹ for isomer II, respectively.

     

Simultaneous HPLC–DAD Determination of Retinol and Eight Vitamin E Isomers in Human Serum

An efficient, high-performance liquid-chromatographic method with diode-array detection (HPLC–DAD) has been established for simultaneous determination of retinol, α, (β + γ), and δ-tocopherols, and α, β, γ, and δ-tocotrienols in human serum. After deproteinization, the target vitamins in serum were extracted with n-hexane and the extract was evaporated under weak nitrogen flow. The residue was redissolved in methanol and the resulting solution was used for HPLC analysis. Retinol acetate and α-tocopherol acetate were used as internal standards. The internal standard calibration curves were linear over the range of 0.010–50.0 µg mL⁻¹, with correlation coefficients >0.999. Mean recoveries of the method were 86.3–110 %, with intra-day and inter-day relative standard deviations less than 12.2 and 14.9 %, respectively. The detection limits of the method ranged from 0.001 to 0. 002 µg mL⁻¹, and the quantification limits ranged from 0.002 to 0.008 µg mL⁻¹. The method was successfully applied to analysis of the target vitamins in 50 human serum samples; all the analytes were detected at concentrations ranging from <0.002–23.0 µg mL⁻¹.

     

Development and Validation of a Chiral Liquid Chromatographic Method for Quantitative Determination of (+)-OTX015 in (−)-OTX015

A simple, rapid and robust enantioselective method was developed and validated for the quantitation of OTX015 enantiomers [(−)-OTX015 and (+)-OTX015] with ultrahigh-performance liquid chromatography (UHPLC) as per ICH guidelines. The active [(−)-OTX015] and inactive [(+)-OTX015] enantiomers were resolved on a Chiralpak-IA column using methanol consisting of 0.1 % diethyl amine at a flow rate of 1.0 mL min⁻¹. The resolution between the enantiomers was found to be more than 3.7 in the optimized method. The developed method was extensively validated and proven to be robust. The calibration curve for (+)-OTX015 showed excellent linearity over the concentration range of 10–100 µg mL⁻¹. The limit of detection and the limit on quantitation for (+)-OTX015 were 5 and 10 µg mL⁻¹, respectively. The recovery for (+)-OTX015 ranged between 100.7 and 102.5 % in the bulk drug sample of (−)-OTX015. The proposed method was found to be suitable and accurate for quantitative determination of (+)-OTX015 in bulk drug.

     

N‐(Substituted benzyl)‐3,5‐bis(benzylidene)‐4‐piperidones: Synthesis and Preliminary Anti‐leukemia Activity (I)

A series of novel N‐(substituted benzyl)‐3,5‐bis(benzylidene)‐4‐piperidones 5a – 5o were synthesized with substituted benzylamines as raw materials via a series of Michael addition, Dieckmann condensation, hydrolysis decarboxylation and aldol condensation. The structures were confirmed by ¹H NMR, IR, MS techniques and elemental analysis. Assay‐based antiproliferative activity study using leukemic cell lines K562 revealed that most of the title compounds have high effectiveness in inhibiting leukemia K562 cells proliferation, among which the compounds 5g (IC₅₀=7.81 µg·mL⁻¹), 5k (IC₅₀=6.35 µg·mL⁻¹), 5l (IC₅₀=7.20 µg·mL⁻¹), and 5o (IC₅₀=5.79 µg·mL⁻¹) have better inhibition activities than standard 5‐fluorouracil (IC₅₀=8.56 µg·mL⁻¹).     

Determination of the Enantiomeric Purity of Trelagliptin by Pre-Column Derivatization and Liquid Chromatography on a Chiral Stationary Phase

A precise and sensitive LC method for determination of enantiomeric purity of trelagliptin has been developed and validated. Pre-column derivatization was performed before separation. Baseline separation with a resolution factor >2.5 was accomplished within 10 min by use of a Chiralpak AD column (250 × 4.6 mm; particle size 5 µm) and n-hexane–2-propanol (90:10 v/v) as mobile phase at a flow rate of 1 mL min⁻¹. Eluted analytes were monitored by UV detection at 260 nm. The effects of mobile phase composition and temperature on enantiomeric selectivity and on resolution of enantiomers were thoroughly investigated. Calibration curves were plotted within the concentration range 0.005–2 mg mL⁻¹ (n = 12), and recoveries between 98.23 and 101.34 % were obtained, with relative standard deviation (RSD) <1.39 %. LOD and LOQ for the trelagliptin derivative were 1.51 and 5.03 µg mL⁻¹; those for its enantiomer were 1.49 and 4.94 µg mL⁻¹, respectively. The method was evaluated and validated by analysis of bulk samples of trelagliptin of different enantiomeric purity. It was demonstrated that the method was accurate, robust, and sensitive, and enabled practical analysis of real samples.

     

The Development and Validation of a Stability-Indicating UHPLC-DAD Method for Determination of Perindopril l-Arginine in Bulk Substance and Pharmaceutical Dosage Form

A stability-indicating ultra-high-performance liquid chromatography (UHPLC) method with a diode array detector was developed and validated for the determination of cis/trans isomers of perindopril l-arginine in bulk substance and pharmaceutical dosage form. The separation was achieved on a Poroshell 120 Hilic (4.6 × 150 mm, 2.7 µm) column using a mobile phase composed of acetonitrile–0.1 % formic acid (20:80 v/v) at a flow rate of 1 mL min^?1. The injection volume was 5.0 µL and the wavelength of detection was controlled at 230 nm. The selectivity of the UHPLC-DAD method was confirmed by determining perindopril l-arginine in the presence of degradation products formed during acid–base hydrolysis and oxidation as well as degradation in the solid state, at an increased relative air humidity and in dry air. The method’s linearity was investigated in the ranges 0.40–1.40 µg mL^?1 for isomer I and 0.40–2.40 µg mL^?1 for isomer II of perindopril l-arginine. The UHPLC-DAD method met the precision and accuracy criteria for the determination of the isomers of perindopril l-arginine. The limits of detection and quantitation were 0.1503 and 0.4555 µg mL^?1 for isomer I and 0.0356 and 0.1078 µg mL^?1 for isomer II, respectively.     

On-Line Two-Dimension Liquid Chromatography for the Analysis of Ingredients in the Medicinal Preparation of Coptis Chinensis Franch

An on-line two-dimension microflow liquid chromatography was developed for better separation and analysis of the highly complex ingredients of medicinal preparation of traditional Chinese medicine Coptis Chinensis Franch. A two-valve switching system was utilized for two-dimension chromatography with strong cation exchange and reverse-phase capillary columns separation. The components were separated well by this system and yielded over 420 peaks. Under the optimal condition, 4 compounds were detected quantitatively. A good linear relationship was obtained from 0.2 µg mL^?1 to 24 µg mL^?1with detection limits (S/N = 3) ranging from 0.05 µg mL^?1 to 0.2 µg mL^?1for the compounds. We demonstrated that the method can be successfully applied to the analysis of a natural complex sample, with satisfactory results.     

Quantitative Analysis of Fudosteine in Human Plasma Using a Simple Precipitation Step Followed by LC–MS–MS

Fudosteine [(-)-(R)-2-amino-3-(3-hydroxypropylthio) propionic acid] is a synthetic derivative of l-cysteine as a novel expectorant. To facilitate pharmacokinetics studies of fudosteine in man, a sensitive and specific LC–MS–MS method for the quantitative detection of fudosteine in human plasma was developed and validated. The method involved the addition of fosfomycin as internal standards, protein-precipitation, HPLC separation, and quantification by MS/MS system using negative electrospray ionization in the multiple reaction monitoring mode. The precursor → product ion transitions were monitored at 177.6 → 90.9 and 136.8 → 78.9 for fudosteine and the IS, respectively. The lower limit of quantitation (LLOQ) was 5 ng mL⁻¹. The calibration curves for fudosteine was linear over a concentration range of 0.005–10 µg mL⁻¹. The intra- and inter-day analyses of QC samples at 0.01, 1, 10 µg mL⁻¹ indicated good precision %RSD between 92.00 and 101.02%. The fudosteine was stable in human plasma stored at room temperature for at least 12 h, 4 °C for at least 12 h, −20 °C for at least 20 days, and at least three freeze–thaw cycles procedures. This accurate and highly specific assay provides a useful method for evaluating the pharmacokinetics of fudosteine in human.

     

Simultaneous Determination and Drug Dissolution Testing of Combined Amlodipine Tablet Formulations Using RP-LC

In the proposed work, the simultaneous analysis of amlodipine–rosuvastatin and amlodipine–atorvastatin in their dosage forms was achieved. Simultaneous dissolution profiles of the amlodipine–rosuvastatin and amlodipine–atorvastatin tablets are realized using Apparatus II with a simple, accurate and precise RP-LC method. The mobile phase consisting of 0.2 % H₃PO₄ and pH 5:methanol:acetonitrile (46:27:27) was used. The samples of 10 µL were injected onto a Zorbax SB C18 (100 mm, 4.6 mm, 3.5 µm particle size) column with 1.2 µL min⁻¹ flow rate. The samples were detected at 236 nm. By plotting peak area ratios vs. concentration, the linearity for amlodipine–rosuvastatin and amlodipine–atorvastatin was determined. With the developed RP-LC method, AML, ROS and ATOR were detected within the range of 0.25–10, 0.5–10 and 0.25–25 µg mL⁻¹, respectively. LOD and LOQ values were also calculated as 0.028, 0.058, 0.021 and 0.095 µg mL⁻¹, 0.195 µg mL⁻¹, 0.070 µg mL⁻¹ for AML, ROS and ATOR, respectively. System suitability tests parameters, such as capacity factor, selectivity to previous peak, selectivity to next peak, resolution to previous peak, resolution to next peak, tailing factor, theoretical number of plates, were performed and found coherent with the ICH guideline parameters. The proposed method has been extensively validated in terms of recovery, and recovery results were between 99 and 101 %. For proving the precision, between-day and within-day repeatability results of the method were proposed. The method can be used for the simultaneous determination of amlodipine–rosuvastatin and amlodipine–atorvastatin.

     

Identification and Quantification of Heterologous Compounds Parthenin and Organic Acids in Parthenium Hysterophorus L. Using HPLC-PDA-MS-MS

Parthenium hysterophorus L., is an obnoxious weed known for its environmental health hazards and medicinal uses. These characteristics are due to presence of sesquiterpene lactones and organic acids; therefore a rapid and sensitive analytical procedure using HPLC-PDA-MS-MS was developed and optimized for separation, identification, and quantification of parthenin and six organic acids. Separation and characterization of compounds was achieved on a RP-C18 column with 1% acetic acid in water (A) and acetonitrile (B) as a mobile phase at a flow rate of 0.6 mL min^?1 and by matching their UV and mass spectra with reference compounds. Six organic acids (ferulic acid, 0.1 mg g^?1 to coumaric acid, 13.6 mg g^?1) and parthenin (27.4 mg g^?1) were characterized within 26 minutes of chromatographic separation in plant extract. The calibration curves are linear with correlation coefficients from 0.985 to 0.998, limit of detection and quantification ranged between 1.0 µg mL^?1 (anisic acid) to 2.2 µg mL^?1 (parthenin) and 2.5 µg mL^?1 (coumaric acid) to 5.2 µ g mL^?1 (parthenin) and recovery ranged between 90.9% to 97.3%. To the best of our knowledge this is the first report for the simultaneous separation of parthenin and organic acids. The method is applicable for screening of commercial crops, medicinal plants, and their products which might be mixed with P. hysterophorus during harvesting period.     

Development and Validation of a Stability-Indicating HPLC Method for Determination of Ciprofloxacin Hydrochloride and its Related Compounds in Film-Coated Tablets

The objective of the current study was the development and subsequent validation of a simple, sensitive, precise and stability-indicating reversed-phase HPLC method for the determination of ciprofloxacin HCl in pharmaceutical dosage forms in the presence of its potential impurities. The chromatographic separation of ciprofloxacin HCl and its related compounds was achieved on an Inertsil ODS3 column using UV detection. The optimized mobile phase consisted of phosphoric acid solution: acetonitril. The proposed method provided linear responses within the concentration range 250–750 μg mL⁻¹ for ciprofloxacin HCl and 0.5–1.5 μg mL⁻¹ for its related compounds. LOD and LOQ values for the active substance were 5.159 and 15.632 μg mL⁻¹, respectively. Correlation coefficients (r) of the regression equations for the impurities were greater than 0.99 in all cases. The precision of the method was demonstrated using intra- and inter-day assay RSD% values which were less than 1% in all instances. No interference from any components of pharmaceutical dosage forms or degradation products was observed.

     

Simultaneous Determination of Andrographolide and Dehydroandrographolide in Chicken Plasma for Application to Pharmacokinetic Studies

A simple, suitable reverse phase liquid chromatographic method was developed for simultaneous determination of andrographolide (1) and dehydroandrographolide (2) in chicken plasma after orally administrating the ultra-fine powder of Andrographis paniculata. Plasma samples were extracted with ethyl acetate. Analysis of the extract was performed on a reversed-phase C18 column with gradient eluent composed of acetonitrile and 0.5% acetic acid. The flow rate was kept at 1 mL min⁻¹ and the detection wavelength was set at 225 and 255 nm for 1 and 2, respectively. All calibration curves showed good linear regression (R ≥ 0.9991). The good precision and recoveries with intra-day and inter-day were 3.2–8.7% and 91.1–98.4%, respectively. The limit of detection was 0.016 µg mL⁻¹ and the limit of quantitation was 0.040 µg mL⁻¹ for the target analytes. This validated method has been successfully applied in the pharmacokinetics study of 1 and 2 after orally administrating the Andrographis paniculata ultra-fine powder to chicken.

     

Simultaneous Determination of Sildenafil,Tadalafil, and Vardenafil in Pharmaceutical Preparations by High-Temperature Gas Chromatography/Mass Spectrometry

Sildenafil (SD), tadalafil (TD), and vardenafil (VD) are drugs used to treat erectile dysfunction (ED). Pharmaceutical counterfeiting-related SD, TD, and VD compounds are becoming a critical problem due to their harmful side-effects. Especially, SD and TD are two major counterfeit ED drugs in South Korea. In this study, a new high-temperature gas chromatography/mass spectrometry (HTGC/MS) method was developed for the rapid determination of SD, TD, and VD in pharmaceutical preparations. A HT GC column was introduced for the rapid analysis of high molecular weight and high boiling point compounds. High-speed centrifugation led to shortened time for results. Calibration curves were linear (R ² ≥ 0.9972) over the concentration ranges of 12.5–100 µg mg⁻¹ for TD and VD, and 25–175 µg mg⁻¹ for SD. The intra- and inter-day precisions were within 7.5 %, while the intra- and inter-day accuracies ranged from –3.0 to 8.0 %. The limits of quantification were 0.7 µg mg⁻¹ (SD and TD) and 0.13 µg mg⁻¹ (VD). The recoveries were in the range of 87.3–102.4 %. The developed method was rapid and accurate both for routine quantitative measurement of SD, TD, and VD in pharmaceutical preparations as well as screening their suspected counterfeit compounds.

     

Development and Validation of the Chiral HPLC Method for Daclatasvir in Gradient Elution Mode on Amylose-Based Immobilized Chiral Stationary Phase

A selective and specific high-performance liquid chromatography method for the determination of daclatasvir enantiomers has been developed and validated. Various immobilized polysaccharide-based chiral stationary phases were used to define a separation strategy utilizing normal-phase and polar organic chromatography modes. Excellent resolution between daclatasvir and its enantiomer was achieved on amylose tris (3-chlorophenylcarbamate) stationary phase, namely CHIRALPAK ID-3, using binary gradient containing acetonitrile:diethylamine and methanol:diethylamine as the mobile phase. The flow rate of the mobile phases was maintained at 1.0 mL min⁻¹ while the column oven temperature was maintained at 40 °C. The column effluent was monitored by UV detection at 315 nm. In comparison with isocratic method, the binary gradient method offered excellent peak shape and improved resolution between daclatasvir and its enantiomer while maintaining the specificity with diastereomers. The method was found to be precise, accurate, and linear (R ² > 0.999). Limit of detection and limit of quantitation of the enantiomer were found to be 0.083 µg mL⁻¹ as and 0.25 µg mL⁻¹, respectively. Recovery of the enantiomer was found to be in the range of 90 to 112 %.

     

Comparison of HPLC Versus HPTLC-Densitometry for the Quantification of Chromone Glucosides in Saposhnikoviae divaricatae Radix

A HPLC and a HPTLC-densitometric method were developed for the quantification of prim-O-glucosylcimifugin and 4′-O-β-d-glucosyl-5-O-methylvisamminol the major chromone glucosides in the roots of Saposhnikovia divaricata. The validation of both methods resulted in comparable parameters regarding stability, specificity, linearity, robustness, precision and recovery, whereas complementary advantages were obtained concerning LOD and LOQ. The HPTLC-based densitometry revealed a lower LOD (1.11 versus 4.37 μg mL⁻¹ in HPLC) and LOQ (3.36 versus 13.24 μg mL⁻¹ in HPLC) for prim-O-glucosylcimifugin, whereas the HPLC resulted in a lower LOD (1.00 versus 4.10 μg mL⁻¹ in HPTLC-densitometry) and LOQ (3.04 versus 12.46 μg mL⁻¹ in HPTLC-densitometry) for 4′-O-β-d-glucosyl-5-O-methylvisamminol. Both methods revealed nearly matching contents of the chromones after analysis of different commercially available batches of Saposhnikoviae divaricatae radix with a total content for both chromone glycosides in the range from 0.31 ± 0.011 to 0.56 ± 0.021 % determined by HPLC and between 0.34 ± 0.011 and 0.61 ± 0.009 % determined by HPTLC. The plant material cultivated in Germany showed a very similar content and ratio of both chromone glucosides in comparison to the standard batches originating from China.

     

HPLC and UPLC Methods for the Simultaneous Determination of Enalapril and Hydrochlorothiazide in Pharmaceutical Dosage Forms

High efficiency and less elution are the basic requirements of high-speed chromatographic separation. In this study, a new gradient reverse phase chromatographic methods were developed using HPLC and UPLC systems for simultaneous determination of enalapril maleate (ENL) and hydrochlorothiazide (HCZ) in pharmaceutical dosage forms. The chromatographic separations of ENL and HCZ were achieved on a Waters μ-Bondapak C 18, (300 × 3.9 mm, 10 μm) and Waters Acquity BEH C18 (100 × 2.1 mm, 1.7 μm) columns for HPLC within 5.30 min and UPLC within a short retention time of 1.95 min, respectively. A linear response was observed over the concentration range 0.270–399 μg mL⁻¹ of ENL, 0.260–399 μg mL⁻¹ of HCZ for HPLC system and 0.270–399 μg mL⁻¹ of ENL and 0.065–249 μg mL⁻¹ of HCZ for UPLC system. Also, limit of detection for ENL was 1.848 ng mL⁻¹ and 31.477 ng mL⁻¹ for HCZ, 2.804 ng mL⁻¹ for ENL and 2.943 ng mL⁻¹ for HCZ using HPLC and UPLC, respectively. The proposed methods were validated according to ICH guideline with respect to precision, accuracy, and linearity. Forced degradation studies were also performed for both compounds in bulk drug samples to demonstrate the specificity and stability indicating power of the HPLC method. Comparison of system performance with conventional HPLC was made with respect to analysis time, efficiency, and resolution.

     

Improved Multianalyte Determination of the Intense Sweeteners Aspartame and Acesulfame‐K with a Solid Sensing Zone Implemented in an FIA Scheme

Abstract

A multianalyte flow‐through sensor is proposed for the simultaneous determination of aspartame (AS) and acesulfame‐K (AK) in tabletop sweeteners. The procedure is based on the transient retention of AK in the ion exchanger Sephadex DEAE A‐25 placed in the flow‐through cell of a monochannel flow injection analysis (FIA) set‐up using pH 2.70 ortophosphoric acid/sodium dihydrogen phosphate buffer 0.06 M as carrier. In these conditions AS is very weakly retained, which makes it possible to measure the intrinsic ultraviolet (UV) absorbance of first AS and then AK after desorption by the carrier itself. The applicable concentration range, the detection limit, and the relative standard deviation were the following: for AS, from 10 to 100 µg mL^?1; 5.65 µg mL^?1; 3.4% (at 50 µg mL^?1); and for AK, between 40 and 100 µg mL^?1; 11.9 µg mL^?1 and 1.61% (at 50 µg mL^?1). The method was applied and validated satisfactorily for the determination of AS and AK blends in tabletop sweeteners. The results were compared against an HPLC reference method.     

Determination of Spectinomycin in Human Urine Using CE Coupled with Electrogenerated Chemiluminescence

A method of capillary electrophoresis with electrogenerated chemiluminescence was developed for the detection of spectinomycin. The linear ranges were from 0.01 to 1.0 mg mL⁻¹ for spectinomycin. The limit of detection for spectinomycin with a signal-to-noise ratio of 3:1 was found to be 4.0 µg mL⁻¹. For practical application perchloric acid was used to eliminate the protein contained in human urine, which is very harmful to electrophoresis separation. The recoveries of spectinomycin at different levels in human urine were between 91.1 and 106.5%. The RSD was between 2.6 and 4.8% (n = 5–6). It was valuable in clinical and biochemical laboratories for monitoring the drug for various purposes.

     

Simultaneous Determination of Mepyramine Maleate,Lidocaine Hydrochloride,and Dexpanthenol in Pharmaceutical Preparations by Partial Least‐Squares Multivariate Calibration

Abstract

A partial least‐squares calibration (PLS) method has been developed for simultaneous quantitative determination of mepyramine maleate (MAM), lidocaine hydrochloride (LIH), and dexpanthenol (DPA) in pharmaceutical preparations. The resolution of these mixtures has been accomplished by using partial least‐squares (PLS‐2) regression analysis of electronic absorption spectral data without prior separation or derivatization. The experimental calibration matrix was constructed with 27 samples. The concentration ranges considered were 2, 3, 4 µg mL^?1 for MAM, 2, 3, 4 µg mL^?1 for LIH, and 8, 10, 12 µg mL^?1 for DPA. The absorbances were recorded between 190 and 340 nm every 5 nm. The results show that PLS‐2 is a simple, rapid, and accurate method applied to the determination of these compounds in pharmaceuticals.