Development and Validation of a New LC Method for Analysis of Brimonidine Tartrate and Related Compounds

A novel liquid chromatographic method for analysis three potential impurities in brimonidine tartrate drug substance has been developed and validated. Efficient chromatographic separation was achieved on a C₈ column (250 mm × 4.6 mm, 5-μm particles) with a simple mobile-phase gradient at a flow rate of 1.0 mL min⁻¹. Quantification was achieved by use of ultraviolet detection at 248 nm. Resolution between brimonidine tartrate and its three potential impurities was greater than 3.0. Regression analysis showed the r value (correlation coefficient) was >0.999 for brimonidine and its three impurities. The method was capable of detecting all three impurities of brimonidine tartrate at levels below 0.07 μg in a test concentration of brimonidine tartrate of 1.0 mg mL⁻¹ and for an injection volume of 10 μL. A solution of brimonidine tartrate in acetonitrile–water 2:8 (v/v) was stable for 48 h. The drug was subjected to stress conditions as prescribed by the ICH. Degradation was found to occur slightly under oxidative stress conditions but the drug was stable to aqueous, acidic, and basic hydrolysis, and photolytic and thermal stress. The assay of the stressed samples was calculated relative to a qualified reference standard and the mass balance was found close to 99.8%. The method was validated for linearity, accuracy, precision, and robustness.

     

Separation of Clinical Grade 188Re from 188W Using Polymer Embedded Nanocrystalline Titania

Currently, ¹⁸⁸Re is obtained from ¹⁸⁸W/¹⁸⁸Re chromatographic generator containing alumina which has a limited capacity (~80 mg Wg⁻¹) for ¹⁸⁸W. This results in high bolus volumes of ¹⁸⁸Re, which often needs to be concentrated before radiolabeling. We have demonstrated the feasibility of using polymer embedded nano crystalline titania (TiP), a novel high capacity sorbent material (~300 mg Wg⁻¹), for developing a ¹⁸⁸W/¹⁸⁸Re chromatographic generator. A TiP based chromatographic ¹⁸⁸W/¹⁸⁸Re generator was developed in which ¹⁸⁸Re could be eluted with 0.9% saline solution. About 90% of the ¹⁸⁸Re could be recovered in the first 4–5 mL of total activity with more than 80% yield. The purity of ¹⁸⁸Re is adequate for clinical applications.

     

Assessment of a Plantibody HB-01 Purification Strategy at Different Scales

Transgenic plant investigations focus on fine-regulation of the recombinant protein expression and purification strategies. In this article, preliminary experiments were done at analytical-scale to decide the best plantibody HB-01 purification strategy. Once it was assumed, the purification efficiency was assessed at different scales (10–600 kg of biomass). The plantibody purity measured by SDS-PAGE and LC-GF was over 90%, yielding 9.9 ± 6.2–18.6 ± 0.9 mg plantibody kg⁻¹ of biomass and 39.9 ± 7.9–48.7 ± 2.1% of recovery. Significant differences were not observed among these parameters at these scales. Plant DNA contents were <3.3 ng mg⁻¹ of plantibody, which is considered very low for the plantibody HB-01 application in the hepatitis B vaccine production.

     

Development of a Reversed-Phase Liquid Chromatographic Assay for the Quantification of Total Persipeptides in Fermentation Broth

The emergence and prevalence of multi-drug-resistant bacterial strains increase the potential for outbreaks of incurable infections. The discovery of novel antibiotics and pharmacological preparations requires the identification of novel bioactive small molecules. A specific, sensitive, and reliable quantification method using high-performance liquid chromatography (HPLC) with UV detection was developed for the determination of total persipeptides (A and B), which are cyclic pentapeptides found in the fermentation broth of Streptomyces zagrosensis UTMC 1154 that exhibit bioactivity against methicillin-resistant Staphylococcus aureus (MRSA). A simple liquid–liquid extraction (LLE) method using butanol was employed to extract persipeptides from the fermentation broth prior to HPLC analysis. The chromatographic separation of persipeptides and the internal standard, virginiamycin, was achieved with a gradient of acetonitrile and water on a C18 reversed-phase analytical column in a 25-min analytical run utilizing a flow rate of 0.8 mL min⁻¹ and detection at 210 nm. The whole assay was validated, and the method presented a linear response range with a regression coefficient of determination R ² of 0.9996 for the quantification of persipeptides in the concentration range of 3.9–250.0 µg mL⁻¹, as well as extraction recoveries ranging from 54.78 ± 9.83 % to 56.45 ± 16.33 %. The bias and the precision of the proposed method were <10 %. The detection and quantification limits for the persipeptides were 27 and 83 µg L⁻¹, respectively.

     

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 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.

     

Stress Degradation Behavior of Entacapone and Development of LC Stability-Indicating Related Substances and Assay Method

A new, sensitive, stability indicating gradient RP-LC related substances and assay method has been developed for the quantitative determination of entacapone in bulk drugs. Efficient chromatographic separation was achieved on a C18 stationary phase with simple mobile phase combination of buffer and acetonitrile. Buffer consisted of 0.1% orthophosphoric acid, delivered in a gradient mode and quantitation was carried out using ultraviolet detection at 220 nm with a flow rate of 1.5 mL min⁻¹. In the developed LC method the resolution (R _s ) between entacapone and its three potential process impurities were found to be >2.0. Regression analysis showed an r ² value (correlation coefficient) >0.99 for entacapone and its three potential impurities. This method was capable to detect all three process impurities of entacapone at a level of 0.003% with respect to test concentration of 0.5 mg mL⁻¹ for a 20 μL injection volume. The inter- and intra-day precision values for all three impurities and for entacapone was found to be within 2.0% RSD. The method has shown good and consistent recoveries for entacapone in bulk drugs (99.2–101.5%) and its three impurities (99.5–102.2%). The test solution was found to be stable in diluent for 48 h. The drug substances were subjected to stress conditions of hydrolysis, oxidation, photolysis and thermal degradation. Considerable degradation was found to occur in acid stress, base stress and oxidative conditions. The stressed test solutions were assayed against the qualified working standard of entacapone and the mass balance in each case was close to 99.7% indicating that the developed method was stability-indicating. The developed RP-LC method was validated with respect to linearity, accuracy, precision and robustness.

     

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.

     

Determination of Three Nonsteroidal Anti-Inflammatory Drugs in Human Plasma by LC Coupled with Chemiluminescence Detection

A simple and sensitive method was developed for the determination of three nonsteroidal anti-inflammatory drugs (NSAIDs)—ibuprofen, naproxen and fenbufen in human plasma. The method involved in column liquid chromatographic separation and chemilumenescence (CL) detection based on the CL reaction of NSAIDs, potassium permanganate (KMnO₄) and sodium sulfite (Na₂SO₃) in sulfuric acid (H₂SO₄) medium. The chromatographic separation was carried out using a reversed-phase C₁₈ column, which allowed the selective determination of the three medicines in the complicated samples. The special features of the CL detector provided lower LOD for determination than that of existing chromatographic alternatives. The results indicated that the linear ranges were 0.01–10.0 μg mL⁻¹ for ibuprofen, 0.001–1.0 μg mL⁻¹ for naproxen, and 0.01–10.0 μg mL⁻¹ for fenbufen. The limits of detection were 0.5 ng mL⁻¹ for ibuprofen, 0.05 ng mL⁻¹ for naproxen and 0.5 ng mL⁻¹ for fenbufen (S/N = 3). All average recoveries were in the range of 90.0–102.3%. Finally, the method had been satisfactorily applied for the determination of ibuprofen, naproxen and fenbufen in human plasma samples.

     

Magnetic Solid Phase Extraction Coupled with HPLC Towards Removal of Pigments and Impurities from Leaf-derived Paclitaxel Extractions of Taxus baccata and Optimization via Response Surface Methodology

A reusable and cost-effective magnetic graphite oxide (Fe₃O₄NPs@GO) nanocomposite was fabricated and applied for pre-purification of paclitaxel from leaf-derived crude extract of Taxus baccata. Furthermore, the potential roles of three crucial criteria (i.e., adsorbent dosage, sorption temperature and agitation/shaking power) on the two responses [i.e., efficiency of plant pigments removal (EPPR) and efficiency of taxol purity (ETP)] were examined and simultaneously optimized through response surface methodology. The nanocomposite was accurately characterized using TEM, AFM, BET, FT-IR, Raman and VSM. Moreover, for both proposed second-degree polynomial regression models, highly significant correlations were achieved between the experimental and predicted data (p < 0.0001). Meanwhile, the optimum conditions to simultaneously acquire the maximum EPPR (94.0 %) and ETP (11.4 %) were recorded as adsorbent dosage of 37.7 g L⁻¹, sorption temperature of 30.7 °C and agitation power of 153.1 rpm; and the predictive results were confirmed using experimental rechecking survey. Interestingly, upon five consecutive treatments, the nanocomposite still exhibited substantial potency in eliminating large amounts of plant pigments and impurities (up to 90 %), without significant reduction on sorption capacity and magnetism thereof. Our results demonstrated that the current nanocomposite, as SPE sorbent for MSPE, could be a simple, fast and reusable approach for HPLC-based purification studies of paclitaxel, and probably other plant secondary metabolites.

     

Stress Degradation Behavior of Desipramine Hydrochloride and Development of Suitable Stability-Indicating LC Method

A simple reverse phase liquid chromatographic method was developed for the quantitative determination of desipramine hydrochloride and its related impurities in bulk drugs which is also stability-indicating. During the forced degradation at hydrolysis, oxidative, photolytic and thermal stressed conditions, the degradation results were only observed in the oxidative stress condition. The blend of the degradation product and potential impurities were used to optimize the method by an YMC Pack Pro C₁₈ stationary phase. The LC method employs a linear gradient elution with the water–acetonitrile–trifluoroacetic acid as mobile phase. The flow rate was 1.0 mL min⁻¹ and the detection wavelength 215 nm. The stressed samples were quantified against a qualified reference standard and the mass balance was found close to 99.0% (w/w) when the response of the degradant was considered to be equal to the analyte (i.e. desipramine). The developed RP-LC method was validated in agreement with ICH requirements.

     

Preparative Electrochromatography of Tea Polyphenols and Caffeine

A novel low-pressure preparative electrochromatography apparatus was set up to implement the separation of small polar compounds. In this apparatus, a distinguished bottom “T”-shape electrode chamber was designed to remove electrolysis gas and meanwhile enable the apparatus to separate small-molecule solutes. Partial separation of the model sample, crude tea extract (mainly containing (−)-epigallocatechin gallate, (−)-epicatechin gallate and caffeine) by hydrophobic macroporous adsorption column (maximum 40 cm × 20 mm ID) with electric field (maximum 111.0 V cm⁻¹) proved the effectiveness of the electrochromatography apparatus. The fact that the total solute recoveries were over 90% showed the qualification of the apparatus for preparative purpose. The stronger the electric field, the more obvious the electrically induced effects. An alternative in-liquid load manner (loading sample in liquid after the electric field was applied) was proposed, which could further enhance the electrically induced effects than in-column load manner (loading sample on resin bed before applying electric field). Scale-up on electrochromatography by column diameter from 6 to 20 mm resulted in similar electrically induced effects on peak resolutions. All of these investigations revealed that the new technology was feasible and promising on separating small polar compounds, for it inherits the advantages of both liquid chromatography and electrophoresis.

     

TLC Determination of Amitriptyline HCl,Trifluoperazine HCl,Risperidone and Alprazolam in Pharmaceutical Products

A simple, sensitive, and precise thin layer chromatographic (TLC) method for simultaneous analysis of psychopharmacological drugs like amitriptyline HCl, trifluoperazine HCl, risperidone and alprazolam in their single dosage forms has been developed, validated, and used for determination of the compounds in commercial pharmaceutical products. The TLC separation was carried out on Merck TLC aluminium sheets of silica gel 60 F254 using carbon tetrachloride:acetone:triethylamine (8:2:0.3, v/v/v), as mobile phase. Densitometric measurements of their spots were achieved at 250 nm over the concentration range for amitriptyline HCl (50–1,200 ng spot⁻¹), trifluoperazine HCl (50–1,200 ng spot⁻¹), risperidone (100–2,400 ng spot⁻¹) and alprazolam (25–600 ng spot⁻¹). Limit of detection (LOD) for amitriptyline HCl (20 ng spot⁻¹), trifluoperazine HCl (20 ng spot⁻¹), risperidone (40 ng spot⁻¹) and alprazolam (5 ng spot⁻¹) was obtained. The study showed that TLC was sensitive and selective for determination of amitriptyline HCl, trifluoperazine HCl, risperidone and alprazolam using a single mobile phase. This proposed method is able for simultaneous determination of psychopharmacological drugs and also applicable for analysis of pharmaceutical formulations.

     

Determination of Repertaxin Enantiomeric Purity by HPLC on Chiral Stationary Phase

A precise and sensitive LC method for the determination of repertaxin enantiomeric purity has been developed and validated. Baseline separation with a resolution higher than 2.0 was accomplished within 20 min using a Chiralpak AD-H 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, temperature and flow rate on enantiomeric selectivity and on resolution of enantiomers were investigated. Calibration curves were plotted within the concentration range between 0.002 and 1.0 mg mL⁻¹ (n = 3), and relative standard deviation (RSD) of the inter-batch assay and intra-batch assay was less than 1.27 and 1.16 %. LOD and LOQ for repertaxin were 0.65 and 2.19 μg mL⁻¹; those for its enantiomer were 0.70 and 2.34 μg mL⁻¹, respectively. The method was evaluated and validated by analysis of bulk samples of repertaxin of different enantiomeric purity. It was demonstrated that the method was accurate, robust, and sensitive, and enabled practical analysis of real samples.

     

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.

     

Isolation of oxidative degradation products of atorvastatin with supercritical fluid chromatography

The isolation of four oxidative degradation products of atorvastatin using preparative high‐performance liquid chromatography applying at least two chromatographic steps is known from the literature. In this paper it is shown that the same four impurities could be isolated from similarly prepared mixtures in only one step using supercritical fluid chromatography. The methods for separation were developed and optimized. The preparation of the mixtures was altered in such a way as to enhance the concentration of desired impurities. Appropriate solvents were applied for collection of separated impurities in order to prevent degradation. The structures of the isolated impurities were confirmed and their purity determined. The preparative supercritical fluid chromatography has proven to be superior to preparative HPLC regarding achieved purity of standards applying fewer chromatographic as well as isolation steps. Copyright © 2015 John Wiley & Sons, Ltd.     

Proximity-based electrochemical biosensor for highly sensitive determination of methyltransferase activity using gold nanoparticle-based cooperative signal amplification

We describe a turn-on electrochemical biosensor for the detection of methyltransferases (MTases) causing DNA adenine methylation. This biosensor is based on insertion, methylation-resistant cleavage, signal enrichment caused by gold nanoparticles (AuNPs), and a signal probe-dragging strategy. A double-stranded DNA (dsDNA) containing identical MTase and methylation-resistant endonuclease (Mbo I) sites was immobilized on the surface of a gold electrode via Au-S covalent binding. The surface was subsequently treated with MTase and Mbo I and then washed. Results revealed that the surface of the electrode contains methylated dsDNA and 12-base nucleotides residual. Depending on biotin-streptavidin interactions that enabled signal probes and nucleotide residue hybridization and AuNP enrichment, a large number of signal probes labeled with ferrocene (Fc) are captured by the electrode. Under optimal conditions, the differential pulse voltammetry signals of Fc tags (at a working voltage of 0.24 V vs. Ag/AgCl) are linearly related to the log of the MTase activity in the 0.1 to 40 U·mL⁻¹ range. The dynamic range extends from 0.05 to 50 U·mL⁻¹, and the limit of detection is 0.024 U·mL⁻¹ (at an S/N ratio of 3). The assay is well reproducible and highly selective. In our perception, this strategy provides a promising approach for simple, sensitive and selective detection of Dam MTase and may be extended to the determination of other MTase by exchanging the corresponding DNA.

Proximity-based electrochemical biosensor for highly sensitive detection of DNA adenine methylation methyltransferase (Dam MTase) activity using methylation-resistant cleavage coupled with gold nanoparticle based cooperative signal amplification.

     

Determination of Heparin in Plasma by HPLC Coupled with Resonance Light Scattering Detection

High performance liquid chromatography coupled with resonance light scattering detection was developed for separation and determination of heparin in plasma. A good chromatographic separation was achieved using an aminex HPX-87H column (300 mm × 7.8 mm, 9 μm) and a mobile phase of 5.0 mmol L⁻¹ H₂SO₄ at the flow rate of 0.5 mL min⁻¹. The enhanced resonance light scattering signals were derived from a large aggregate formation between heparin and cetyltrimethylammonium bromide used as the molecular recognition probe. The parameters of the post-column reaction (pH, concentration and flow rate of the reagent, length of the reaction coil, and temperature) were optimized. The limit of detection for heparin in plasma was 0.2 mg L⁻¹. The method has been applied to the determination of heparin in dialysis patient plasmas.

     

Characterization of Nineteen Impurities in Roxithromycin by HPLC/TOF and Ion Trap Mass Spectrometry

Nineteen impurities in roxithromycin drug substance made in China were separated and identified by HPLC–MSⁿ (TOF and TRAP) for the further improvement of official monographs in Pharmacopoeias. The fragmentation patterns and structural assignment of these impurities were studied. The column was Shim VP-ODS (250 × 4.6 mm, 5 μm). The mobile phase was 10 m mol L⁻¹ ammonium acetate and 0.1 % formic acid aqueous solution-acetonitrile (62.5:37.5). In positive mode, full scan LC–MS was first performed to obtain the m/z value of the protonated molecules and formulas of all detected peaks on Agilent 6538Q TOF high resolution mass spectrometer. LC–MS-MS and LC–MS-MS–MS were then carried out on the compounds of interest on AB SCIEX 4000 Q TRAP™ composite triple quadrupole/linear ion trap tandem mass spectrometer. The complete fragmentation patterns of nineteen impurities were studied and used to obtain information about the structures of these impurities. The structures of nineteen impurities in roxithromycin drug substance were deduced based on the HPLC–MSⁿ data, in which nine impurities were novel impurities.

     

Development and Validation of an Stability Indicating RP-LC Method for Determination of Imatinib Mesylate

A new, sensitive and stability indicating liquid chromatographic method has been developed for the determination of imatinib mesylate (IM). Efficient chromatographic separation was achieved using a C₁₈ column with simple mobile phase combination delivered in an isocratic mode and quantitation was carried out using ultraviolet detection. For the first time, a novel microwave assisted degradation procedure was employed for stress testing studies. In addition, orthogonal separation technique was applied to demonstrate selectivity of the proposed method. The method has demonstrated excellent linearity over the range of 25–1,600 ng mL⁻¹. Moreover, the method was found to be sensitive with a low limit of detection (3.35 ng mL⁻¹) and limit of quantitation (10.16 ng mL⁻¹). The method has shown good and consistent recoveries (99.35–100.69%) with low intra- and inter-day relative standard deviation (RSD) (<2.5%). Experimental design confirmed that peak area was unaffected by small changes in critical factors, in robustness study. The validated method was successfully applied for determination of IM in pharmaceutical formulations.