Liquid Chromatographic Assay Research Articles

Rapid and sensitive determination of levofloxacin in microsamples of human plasma by high‐performance liquid chromatography and its application in a pharmacokinetic study

A rapid, sensitive and simple high-performance liquid chromatographic assay with ultraviolet detection was developed for the quantification of levofloxacin in microsamples (100 μL) of human plasma. The extraction procedure included a protein precipitation technique and a short chromatographic running time (4.5 min). Analyses were carried out on a Symmetry C18 column using a mixture of acetonitrile and 0.01 m potassium dihydrogen aqueous solution (pH 3.4; 14:86 v/v) as mobile phase. The method provided specificity and was linear (r ≥ 0.9992) over the concentration range 0.1-12 µg/mL. The average absolute recovery was 93.59%. The intra- and inter-day coefficients of variation were <6%. Additionally, levofloxacin was stable in all evaluations. The usefulness of this method was demonstrated in a pharmacokinetic study of levofloxacin in healthy adult volunteers. The present method offers two main advantages: (a) the use of microsamples reduces the total volume of blood to be collected from patients; and (b) it provides a good cost-effectiveness ratio. It is concluded that the method is rapid, simple, sensitive, economical and suitable for the determination of levofloxacin in human plasma using a small volume of sample.

Facile LC-UV methods for simultaneous monitoring of ciprofloxacin and rosuvastatin in API, formulations and human serum.

An efficient, selective and cost-effective liquid chromatographic assay was developed and validated for the simultaneous quantification of ciprofloxacin and rosuvastatin in Active Pharmaceutical Ingredients (API), pharmaceutical formulations and in human serum. The chromatographic system consisted of mobile phase methanol-water, 90:10 v/v at pH 3.0 adjusted with o-phosphoric acid, pumped at 1.0 mL/min through a prepacked Purospher Star C18 (5 µm, 25 × 0.46 cm) column and effluent was monitored at the isosbestic point (255 nm) as well as at the λmax of individual drugs (243 and 271 nm). The method was validated over a linear concentration range of 0.25-15 µg/mL for ciprofloxacin and 0.33-20 µg/mL for rosuvastatin (r(2) ≥ 0.999). The ranges of reliable response (limits of detection and quantitation) for ciprofloxacin were 3-15 and 9-45 ng/mL and 17-29 and 52-88 ng/mL, respectively, for rosuvastatin in all API, pharmaceutical formulations and human serum. Analytical recovery from human serum was >98% and relative standard deviation (RSD) was <2. The accuracies were 97.13-102.55 and 97.41-101.31% and precisions in RSD were 0.04-1.90 and 0.02-1.23% for ciprofloxacin and rosuvastatin, respectively. No matrix interferences, ion suppression/enhancement and carry-over were detected. The total assay run time was less than 5 min. In another study, for optimum performance the detector was programmed for multiwavelength scanning at the absorption maxima of each component. Consequently, the linearity range was improved and limit of detection and quantitation values were down to 1-4 and 4-12 ng/mL for ciprofloxacin and 3-5 and 9-15 ng/mL for rosuvastatin, respectively. The validation parameters fitted ICH guidelines through the isosbestic and individual λmax approach. The small sample volume and simplicity of preparation make this method suitable for use in human serum samples, pharmaceutical formulations, quality control, drug-drug interaction studies, clinical laboratories, drug research centers and forensic medical centers.

Mass spectrometry based identification of geometric isomers during metabolic stability study of a new cytotoxic sulfonamide derivatives supported by quantitative structure-retention relationships.

A set of 15 new sulphonamide derivatives, presenting antitumor activity have been subjected to a metabolic stability study. The results showed that besides products of biotransformation, some additional peaks occurred in chromatograms. Tandem mass spectrometry revealed the same mass and fragmentation pathway, suggesting that geometric isomerization occurred. Thus, to support this hypothesis, quantitative structure-retention relationships were applied. Human liver microsomes were used as an in vitro model of metabolism. The biotransformation reactions were tracked by liquid chromatography assay and additionally, fragmentation mass spectra were recorded. In silico molecular modeling at a semi-empirical level was conducted as a starting point for molecular descriptor calculations. A quantitative structure-retention relationship model was built applying multiple linear regression based on selected three-dimensional descriptors. The studied compounds revealed high metabolic stability, with a tendency to form hydroxylated biotransformation products. However, significant chemical instability in conditions simulating human body fluids was noticed. According to literature and MS data geometrical isomerization was suggested. The developed in sillico model was able to describe the relationship between the geometry of isomer pairs and their chromatographic retention properties, thus it supported the hypothesis that the observed pairs of peaks are most likely geometric isomers. However, extensive structural investigations are needed to fully identify isomers’ geometry. An effort to describe MS fragmentation pathways of novel chemical structures is often not enough to propose structures of potent metabolites and products of other chemical reactions that can be observed in compound solutions at early drug discovery studies. The results indicate that the relatively non-expensive and not time- and labor-consuming in sillico approach could be a good supportive tool assisting the identification of cis-trans isomers based on retention data. This methodology can be helpful during the structural identification of biotransformation and degradation products of new chemical entities - potential new drugs.

Bioanalytical Method Development and Validation for Metoprolol Succinate and Telmisartan Using UV Spectro-Photometry and RP-HPLC

An accurate, precise and reproducible bioanalytical UV-spectrophotometric and liquid chromatographic assay method were developed and validated for the determination of metoprolol succinate (MET) and telmisartan (TEL) in blood plasma using protein precipitation technique. Spectrophotometric estimation was done by simultaneous equation method followed by rst order derivative method using 50% methanol as solvent. In this method λ max for MET and TEL were selected at 240.5 nm and 237.5 nm respectively. RP-HPLC analysis was carried out using Prontosil C-18 column (4.6 x 250 mm, 5 μ particle size) and mobile phase composed of methanol:acetonitrile:phosphate buffer (pH-5) in ratio of 35:35:30 v/v/v, at a ow rate of 1.0 mL min -1 and chromatogram was recorded at 225 nm. Linearity of drugs for blood plasma was evaluated over the concentration range of 5-25 μg mL -1 and 8-40 μg mL -1 for MET and TEL in both UV spectrophotometric and RP-HPLC method (the value of r 2 = 0.999 by both the methods for MET and TEL). The developed methods were validated according to ICH guidelines and values of accuracy, precision and other statistical analysis were found to be in good accordance with the prescribed values. Therefore, both the methods can be used for routine monitoring of MET and TEL in industry in the assay of bulk drug, in plasma and tablets.

Improved protein deproteinization method for the determination of meloxicam in human plasma and application in pharmacokinetic study.

A simple, rapid, specific and reliable high-performance liquid chromatographic assay of meloxicam in human plasma has been developed using a C18 reversed-phase analytical column. Reversed-phase chromatography was conducted using a mobile phase of 0.02 potassium dihydrogen phosphate (adjusted to pH 2.7 with phosphoric acid)-acetonitrile-triethylamine (35:65:0.05, v/v) with UV detection at 354 nm. The drug in human plasma was deproteinized using a combination of methanol and chloroform. This method is simple, rapid and consistent with a high recovery of meloxicam in human plasma ranging from 93.29 to 111.09%. Regression analysis for the calibration plot for plasma standards obtained for the drug concentrations between (25-4000) ng/mL indicated excellent linearity (r ≥ 0.9997). The proposed method was applied to study the bioequivalence between Mobic (original) and Melocam (generic) products. The study was conducted on using two tablets (4 × 7.5 mg) of each of the commercial product and the reference standard in a two-way open randomized crossover design involving 20 volunteers. Area under the concentration-time curve, peak concentration (C(max)) and time to reach C(max) were 72,868.61 ng h/mL, 2133.93 ng/mL and 4.06 h for Mobic, and 78,352.52 ng h/mL, 2525.18 ng/mL and 3.61 h for Melocam. Two C(max) were discovered in the pharmacokinetic profiles which confirm enterohepatic recirculation.

A Stability-Indicating High Performance Liquid Chromatographic Assay for the Simultaneous Determination of Pyridoxine, Ethionamide, and Moxifloxacin in Fixed Dose Combination Tablets

Stability indicating reversed phase HPLC method was developed and validated for the simultaneous quantitation of antitubercular drugs, ethionamide (ETH), and moxifloxacin (MOX) with commonly coprescribed vitamin, pyridoxine (PYR) in tablet dosage form. The method was found rapid, precise and accurate. The separation was performed in Hibar 150-4.6, Purospher STAR, RP-18e (5 μm) column, using mobile phase A (0.03 M sodium citrate adjusted to pH 5 with glacial acetic acid) and mobile phase B (100% methanol), ran at variable proportions at flow rate of 1.0 mL/min. The detection was carried out at 320 nm. The method was observed linearly in the range of 2.5–17.5 μg/mL for PYR, 25–175 μg/mL for ETH, and 40–280 μg/mL for MOX with respective limits of detection/quantitation of 0.125 μg/mL/1.28 μg/mL, 0.25 μg/mL/2.56 μg/mL, and 0.35 μg/mL/3.65 μg/mL. The drugs were also subjected to oxidative, hydrolytic, photolytic, and thermal degradation; the degradation products showed interference with the detection of PYR, ETH, and MOX. The proposed method was observed to be effective to quantitate MOX (400 mg), ETH (250 mg), and PYR (25 mg) in fixed dose combination tablet formulation.

Stability of penicillin G sodium diluted with 0.9% sodium chloride injection or 5% dextrose injection and stored in polyvinyl chloride bag containers and elastomeric pump containers

The stability of penicillin G sodium solutions stored in polyvinyl chloride (PVC) bags or elastomeric pump containers was studied. Test samples were prepared by diluting powdered penicillin G sodium (10 million units/10-mL vial) to solutions of 2,500 or 50,000 units/mL with 0.9% sodium chloride injection or 5% dextrose injection. The preparations were transferred to 250-mL PVC bags and elastomeric pump containers. All samples were prepared in triplicate and stored at 5°C. Chemical stability was measured by a stability-indicating high-performance liquid chromatographic (HPLC) assay and by pH evaluation. Particulate matter was evaluated according to compendial standards using a light-obscuration particle count test. Preparations were visually examined throughout the study. After 21 days of storage, all test samples remained chemically stable, with an HPLC assay recovery value of more than 90% of the initial value. After 28 days, all samples prepared with either diluent and stored in PVC bags, as well as the samples diluted to 2,500 units/mL with sodium chloride injection and stored in elastomeric pump containers, did not meet the recovery acceptance limit. For all test samples, the mean pH consistently decreased during storage, from about 6.4 to about 5.5. Particle counts remained acceptable throughout the study, and no change in appearance was observed. Penicillin G for injection (2,500 and 50,000 units/mL) diluted in 0.9% sodium chloride injection or 5% dextrose injection and stored at 5°C in PVC containers or elastomeric pump containers was physically and chemically stable for a period of at least 21 days.

Highly purified mussel adhesive protein to secure biosafety for in vivo applications.

BackgroundUnique adhesive and biocompatibility properties of mussel adhesive proteins (MAPs) are known for their great potential in many tissue engineering and biomedical applications. Previously, it was successfully demonstrated that redesigned hybrid type MAP, fp-151, mass-produced in Gram-negative bacterium Escherichia coli, could be utilized as a promising adhesive biomaterial. However, purification of recombinant fp-151 has been unsatisfactory due to its adhesive nature and polarity which make separation of contaminants (especially, lipopolysaccharide, a toxic Gram-negative cell membrane component) very difficult.ResultsIn the present work, we devised a high resolution purification approach to secure safety standards of recombinant fp-151 for the successful use in in vivo applications. Undesirable impurities were remarkably eliminated as going through sequential steps including treatment with multivalent ion and chelating agent for cell membrane washing, mechanical cell disruption, non-ionic surfactant treatment for isolated inclusion body washing, acid extraction of washed inclusion body, and ion exchange chromatography purification of acid extracted sample. Through various analyses, such as high performance liquid chromatographic purity assay, limulus amoebocyte lysate endotoxin assay, and in vitro mouse macrophage cell tests on inflammation, viability, cytotoxicity, and apoptosis, we confirmed the biological safety of bacterial-derived purified recombinant fp-151.ConclusionsThrough this purification design, recombinant fp-151 achieved 99.90% protein purity and 99.91% endotoxin reduction that nearly no inflammation response was observed in in vitro experiments. Thus, the highly purified recombinant MAP would be successfully used as a safety-secured in vivo bioadhesive for tissue engineering and biomedical applications.

Assay Method for Quality Control and Stability Studies of a New Antidiabetic Agent (S-001-469)

In the present study, for the first time, HPLC method of CDRI compound S-001-469 is reported, S-001-469 is a new potential substituted chalcone based antidiabetic candidate drug under preclinical development phase. The current study illustrates comprehensive stress testing of S-001-469 under different prescribed ICH stress conditions. A reverse phase high performance liquid chromatographic (HPLC) assay method for determination of S-001-469 with its starting materials, intermediates and degradation product has been developed and validated for in process quality control and stability studies. The analytical performance parameters such as linearity, precision, accuracy, specificity, limit of detection (LOD) and lower limit of quantification (LLOQ) were determined according to International Conference on Harmonization guidelines. Compound S-001-469 was subjected to acidic, alkaline, oxidative (H2O2 and photolytic degradations. HPLC separation was achieved on SunFire™ C-18 column (250 mm, 4.6 mm, 5 m, Waters) using a mobile phase consisting of acetonitrile-0.01 M sodium acetate buffer (pH 4.75) (60:40, v/v) at a flow rate of 1 ml/min and the eluents were monitored by photo diode array detector at 360 nm. Based on signal to noise ratio of 3 and 10 the LOD of S001 469 was 0.98 g/ml, while the LLOQ was 3.9 g/ml. The calibration curve was linear in the range of 3.9–125 g/ml.

Development and Validation of Stability-Indicating Liquid Chromatographic Assay for Rifaximin (An Antibiotic) in Bulk and Pharmaceutical Dosage Forms

An isocratic reversed-phase high-performance liquid chromatographic method was developed and validated for the determination of Rifaximin. Chromatographic separation was achieved on a C18 column using an aqueous tetra butyl ammonium hydrogen sulphate (10 mM) (pH 3.37): acetonitrile (40:60, v/v), with flow rate 1.2 mL/min (UV detection at 441 nm). Linearity was observed in the concentration range of 0.1-200 μg/mL (R 2 = 0.9999). The limit of quantitation was found to be 0.0794 μg/mL and the limit of detection was found to be 0.0241 μg/mL. Rifaximin was subjected to stress conditions of degradation in aqueous solutions including acidic, alkaline, oxidation, photolysis and thermal degradation. The forced degradation studies were performed by using HCl, NaOH, H2O2, thermal and UV radiation. Rifaximin is more sensitive towards acidic conditions in comparison to oxidation and very much resistant towards alkaline, thermal and photolytic degradations. The method was validated as per ICH guidelines. The method is simple, specific, precise, robust and accurate for the determination of Rifaximin in pharmaceutical formulations.

A PRIMER EXTENSION DENATURING HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY METHOD FOR THE IDENTIFICATION OF THREE ABCC2 GENETIC POLYMORPHISMS

Background. A number of single nucleotide polymorphisms have been described in the ABCC2 gene that alters drug disposition. The aim of the study was to develop a primer extension denaturing high-performance liquid chromatography (PE-dHPLC) assay to determine three common variants of the ABCC2 gene in the promoter region (−24C > T), exon 10 (1249 G > A), and exon 28 (3972C > T). Methods. Polymerase chain reactions (PCRs) were used to isolate the area of interest in the ABCC2 gene. A comparison of the PCR product was performed between sequencing and dHPLC. Results. A 100%identity match was achieved between groups and allowed for a quick and accurate method to determine three single nucleotide polymorphisms in a single extension reaction. This assay is the first of its type to determine three ABCC2 variants by dHPLC.

High‐performance liquid chromatography assay using ultraviolet detection for urinary quantification of milrinone concentrations in cardiac surgery patients undergoing cardiopulmonary bypass

An analytical assay using liquid-liquid extraction and high-performance liquid chromatography with ultraviolet detection was developed for the quantification of total (conjugated and unconjugated) urinary concentrations of milrinone after the inhalation of a 5 mg dose in 15 cardiac patients undergoing cardiopulmonary bypass. Urine samples (700 μL) were extracted with ethyl-acetate and subsequently underwent acid back-extraction before and after deconjugation by mild acid hydrolysis. Milrinone was separated on a strong cation exchange analytical column. The mobile phase consisted of a constant mixture of acetonitrile:tetrahydrofurane-NaH2 PO4 buffer (40:60 v/v, pH 3.0). Thirteen calibration curves were linear in the concentration range of 31.25-4000 ng/mL, using olprinone as the internal standard (r(2) range 0.9911-0.9999, n = 13). Mean milrinone recovery and accuracy were respectively 85.2 ± 3.1% and ≥93%. Intra- and inter-day precisions (coefficients of variation) were ≤5% and ≤8%, respectively. Over a 24 h collection period, the cumulative urinary milrinone recovered from 15 patients was 26.1 ± 7.7% of the nominal 5 mg dose administered. The relative amount of milrinone glucuronic acid conjugate was negligible in the urine of patients undergoing cardiopulmonary bypass This method proved to be reliable, specific and accurate to determine the cumulative amount of total milrinone recovered in urine after inhalation.

Pharmacokinetics of dronedarone in rat using a newly developed high-performance liquid chromatographic assay method.

A sensitive and selective high-performance liquid chromatographic method for the determination of dronedarone in rat plasma was developed. Dronedarone was extracted using one-step liquid-liquid extraction. The separation of dronedarone was accomplished using a C18 analytical column. The mobile phase was composed of a combination of monobasic potassium phosphate and acetonitrile. The UV detection was at 254 nm for ethopropazine, the internal standard, and after its elution, changed to 290 nm for dronedarone detection. The total analytical run time was 20 min. Mean recovery was >80%; the assay had excellent linear relationships (>0.999) between peak height ratios and plasma concentrations; the lower limit of quantification 25 was ng/mL, based on 100 μL of rat plasma. Accuracy and precision were <18% over the concentration range of 25-500 ng/mL. The assay was applied successfully to the measurement of dronedarone plasma concentrations in rats given the drug orally.

Comparison of a new microbiological assay with a standard high-performance liquid chromatographic method for determination of vitamin B6 in serum.

A high-performance liquid chromatographic (HPLC) assay for vitamin B6 in human serum was compared with a novel microbiological assay (ID-Vit) that uses microtitre plates precoated with a specific microorganism, thus avoiding numerous problems associated with the use of stock cultures utilized by common other microbial assay mit B6. Data obtained using HPLC were compared with 1D-Vit results in 170 healthy individuals and in 68 patients with coronary artery disease (CAD, 37 with acute coronary syndrome [ACS], 31 with stable CAD). Regression and Bland-Altman analysis were performed. Homocysteine in CAD patients was measured by HPLC. The ID-Vit assay correlated well with the HPLC assay (Pearson's r = 0.89 [p < 0.0001] in healthy and 0.82 [p < 0.001] in CAD individuals). Bland-Altman analyses revealed good agreement between the results of both methods in both cohorts, with > or = 95% of all values grouping within the lines of agreement. In CAD patients, mean homocysteine values did not differ between stable CAD and ACS and were normal. Thirty-seven percent of CAD patients had estimated glomerular filtration rates (GFR) below 60 mL/min/1.73m2. GFR correlated inversely with homocysteine levels (r = -0.80, p < 0.001) whereas neither HPLC nor ID-Vit values for B6 did. ID-Vit assay and the HPLC standard are in very good agreement. The new assay can easily be automated and is less laborious than common microbiological assays. The lack of correlation between B6 vitamin and homocysteine can be accounted for by the fact that mean vitamin B6 in our CAD patients was in the normal range and that a relevant percentage of patients had chronic renal disease.

Derivative spectrophotometric and micellar high performance liquid chromatographic methods for simultaneous determination of silymarin and dimethyl-4,4′-dimethoxy-5,6,5′,6′-dimethylene dioxybiphenyl-2,2′-dicarboxylate (DDB) in raw materials and capsules

First derivative ultraviolet spectrophotometric and micellar high performance liquid chromatographic assay methods were developed and validated for the simultaneous determination of silymarin and dimethyl-4,4′-dimethoxy-5,6,5′,6′-dimethylene dioxybiphenyl-2,2′-dicarboxylate (DDB) in the pure form and in combined capsules. The first derivative (D1) was performed by measuring the amplitudes at 279 and 255 nm for silymarin and DDB, respectively. The calibration graphs were linear in the range of 3–40 μg mL−1 for silymarin and 3–30 μg mL−1 for DDB. An isocratic reversed phase chromatographic assay method was also developed for the determination of both drugs, a C18 column (250 mm × 4.6 mm i.d., 5 μm particle size) was used with a mobile phase composed of 0.17 M sodium dodecyl sulphate, 12% butanol and 0.3% triethylamine, all prepared in 0.02 M phosphoric acid and adjusted to pH 3.5. The flow rate was 1 mL min−1. The effluents were monitored at 288 nm and floctafenine was used as the internal standard. Plots of the peak area ratios of each drug to floctafenine were found to be linear in the range of 1–15 μg mL−1 for silymarin and 1–12 μg mL−1 for DDB. The proposed methods were validated according to ICH guidelines. The methods were successfully applied in the estimation of silymarin and DDB in their combined capsules dosage form.

Determination of reduced glutathione, cystein and total thiols in pine pollen powder by in situ derivatization

Glutathione (GSH), cystein (CySH) and total thiols in pine pollen powder were determined by spectrometric, fluorometric and high performance liquid chromatographic (HPLC) assays utilizing 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB, Ellman's reagent) and o-phthaldehyde (OPA) as derivatizing agents. The pollen samples were extracted with phosphate buffer solutions (PBS) containing excess derivatizing agents concomitant with ultrasonic cell rupture. In this way, thiol groups in the samples could be effectively blocked by in situ formation of relatively stable derivatizing products just after the pollen cell ruptured, thereby effectively preventing thiol groups from oxidation during sample preparation and making it possible to perform sensitive and accurate detections. Using a precolumn derivatization method, DTNB, GSH and CySH, as well as other thiol compounds can be determined or estimated by the HPLC method. Total thiols in the samples were determined by the spectrometric method using DTNB as the derivatizing agent. GSH was also selectively determined by the fluorometric method using OPA as the derivatizing agent. The detection results indicated that abundant thiol compounds, such as GSH, CySH and thiol protein, existed in pine pollen powder with measured amounts of 0.50–0.53μmol∙g−1 of GSH, 0.41μmol∙g−1 of CySH and 4.15μmol∙g−1 of total thiols.

Characterization and Determination of Acetyl-Coenzyme a Carboxylase (ACCase) from Eukaryotic Alga Chlorella Sp. USTB-01

In the present study, the Acetyl-coenzyme A carboxylase (ACCase) of Chlorella sp. USTB-01 purified and characterized. Molecular weight of ACCase was calculated to be about 456 kD and was comprised of two identical subunits of 229 kD. The enzymatic characteristics isoelectric point was 6.7. Moreover, a rapid and sensitive assay of High performance liquid chromatography (HPLC) was established to measure overall activity of ACCase in low protein content condition by detect the ACCase activity by measuring ADP production and Acetyl-coenzyme A (Acetyl-CoA).

HPLC DETERMINATION OF DRDE-07 IN MICE PLASMA: PROBABLE ANTIDOTE FOR SULFUR MUSTARD TOXICITY

DRDE-07 is a new synthetic compound found to be the most prophylactic agent against sulfur mustard toxicity. In this study, the development and validation of a high-performance liquid chromatography (HPLC) assay for determination of DRDE-07 concentration in mice plasma for pharmacokinetic studies is described. A simple protein precipitation procedure by ice cold acetone was applied to extract DRDE-07 from plasma. The intra-day and inter-day precision of DRDE-07 ranged from 1.75% to 4.38% and from 1.07% to 3.21%, respectively. Similarly, the intra-day and inter-day accuracy of DRDE-07 ranged from 95.56% to 100.07% and from 95.69% to 101.02%, respectively. The extraction recovery of DRDE-07 was 96.87 ± 1.89% at 1.0 µg/ml (n = 3), 96.43 ± 1.41% at 10 µg/mL (n = 3), and 88.26 ± 1.49% at 100 µg/mL (n = b3). Calibration curves of DRDDE-07 were linear in the concentration range of 1–100 µg/mL in plasma. The limit of quantification was 1 µg/mL. This method was applied to determine DRDE-07 concentration in mice plasma samples for a pharmacokinetic study.

Physico-chemical stability of eribulin mesylate containing concentrate and ready-to-administer solutions

The aim of this study was to determine the stability of commercially available eribulin mesylate containing injection solution as well as diluted ready-to-administer solutions stored under refrigeration or at room temperature. Stability was studied by a novel developed stability-indicating reversed-phase high-performance liquid chromatography (RP-HPLC) assay with ultraviolet detection (detection wavelength 200 nm). Triplicate test solutions of eribulin mesylate containing injection concentrate (0.5 mg/mL) and with 0.9% sodium chloride solution diluted ready-to-administer preparations (0.205 mg/mL eribulin mesylate in polypropylene (PP) syringes, 0.020 mg/mL eribulin mesylate in polypropylene/polyethylene (PE) bags) were stored protected from light either at room temperature (25) or under refrigeration (2-8). Samples were withdrawn on day 0 (initial), 1, 3, 5, 7, 14, 21 and 28 of storage and assayed. Physical stability was determined by measuring the pH value once a week and checking for visible precipitations or colour changes. The stability tests revealed that concentrations of eribulin mesylate remained unchanged over a period of 28 days irrespective of concentration, container material or storage temperature. Neither colour changes nor visible particles have been observed. The pH value varied slightly over time but remained in the stability favourable range of 5-9. Eribulin mesylate injection (0.5 mg/mL) is physico-chemically stable over a period of 28 days after first puncture of the vial. After dilution with 0.9% NaCl vehicle solution, ready-to-administer eribulin mesylate injection solutions (0.205 mg/mL in PP syringe) and infusion solutions (0.02 mg/mL in prefilled PP/PE bags) are physico-chemically stable for a period of at least four weeks either refrigerated or stored at room temperature. For microbiological reasons storage under refrigeration is recommended.

Distribution of Two Bioactive Compounds in Flowers of Trollius chinensis

A comprehensive procedure was established, which combined a high-performance liquid chromatography (HPLC) assay for the simultaneous quantification of 2″-O-β-L-galactopyranosylorientin (OGA) and veratric acid and a gravimetric analysis for the determination of the mass fraction of the floral parts (calyx, corolla, stalk, stamens and pistils and ovary) of Trollius chinensis, to investigate the distribution and identify the enriched floral part(s) of these two compounds in the flowers. The calculated mean distributions of OGA in calyx, corolla, stamens and pistils, stalk and ovary were 83.62, 7.76, 4.35, 2.92 and 1.35%, respectively, whereas those of veratric acid in the corresponding floral parts were 46.41, 9.01, 18.41, 4.11 and 22.06%, respectively, indicating the uneven and noncorresponding distribution of these two compounds. This study extends the application of the HPLC assay and favors the production of OGA and veratric acid from the flowers of T. chinensis in addition to the benefits of breeding, cultivation and utilization of these flowers.