Solvent Gradient Elution Research Articles

Development and validation of an analytical method using liquid chromatography-tandem mass spectrometry for the therapeutic drug monitoring of seven cardiovascular drugs in clinical usage

Therapeutic drug monitoring (TDM) of cardiovascular drugs is essential to improve treatment efficacy and minimize toxicity because of the usage of multiple drugs with a very limited therapeutic range and the high pharmacokinetic variation in patients. We developed and validated a reliable and economical liquid chromatography/tandem mass spectrometry (LC-MS/MS) method for the determination of seven cardiovascular drugs—procainamide, lidocaine, quinidine, deslanoside, digoxin, atorvastatin, and digitoxin—for clinical usage. Serum samples were prepared by simple protein precipitation with an organic solvent consisting of acetonitrile and methanol (2:1 v/v) and analyzed under optimized LC-MS/MS conditions. The chromatographic separations were accomplished within 15 min on a reversed-phase C18 column with a gradient elution of aqueous solvent and acetonitrile while maintaining 0.1 (v/v) % formic acid and 2 mM ammonium formate. The optimized MS/MS conditions in ESI-positive mode offered sufficient sensitivity for the seven cardiovascular drugs (LOQs between 0.5 and 1 ng/mL). This method was fully validated including linearity, selectivity, accuracy, precision, carry-over, and matrix effects. Additionally, stability under several conditions was tested to determine how to handle the standard solutions and serum samples. The seven cardiovascular drugs, simultaneously, were precisely and accurately analyzed in intra- and inter-day assays (RSD < 6 % and recovery between 96.3 and 102.8 %) using only two isotope-labeled internal standards (lidocaine-(diethyl-d10) and digoxin-21, 21, 22-d3). The presented method also showed good accuracy in analyzing the seven drugs in hyperlipidemia, hyperalbuminemia, and hyperglycemia serum, allowing it to be recommended as a common and routine analysis method for cardiovascular drugs in clinical practice.

Development of a rapid and validated stability-indicating UPLC-PDA method for concurrent quantification of impurity profiling and an assay of ipratropium bromide and salbutamol sulfate in inhalation dosage form

The objective of the proposed work was to develop a rapid and new reverse phase ultra-performance liquid chromatographic (RP-UPLC) method for the simultaneous quantification of related impurities of ipratropium bromide and salbutamol sulfate in the combined inhalation dosage form. Herein, the chromatographic separation was achieved on Acquity BEH C18 (100mm×2.1mm, 1.7μm) column by following gradient elution of solvent A as 2mM potassium dihydrogen phosphate with 0.025% of 1-pentane sulphonic acid sodium salt (pH 3.0 buffer) and solvent B as pH 3.0 buffer, acetonitrile and methanol in the ratio of (32:50:18, v/v/v) at a flow rate of 0.3mL/min. The samples were detected and quantified at 220nm. To prove the stability-indicating potential of the method, forced degradation studies were performed using acidic, basic, oxidative, thermal, and photolytic conditions. After sufficient exposure, the resultant solutions were injected and found that all degradants and impurities formed during stress studies were well separated from each other and from the main peak compounds. The performance of the method was validated according to the present ICH Q2 (R1) guidelines. The method has good linearity (r≥0.999) and consistent recoveries were obtained with a range of 91.3-108.8% for all compounds. The % RSD obtained for the precision experiments was less than 5% and also there is a good sensitivity (LOQ≤0.5μg/mL) for all compounds. The intended method proved its applicability and that it can be beneficial to pharmaceutical industries for quick quantification of related impurities and assay in quality control department for analysis of ipratropium bromide and salbutamol sulfate inhalation dosage form.

A high throughput method for Monitoring of Sorafenib, regorafenib, cabozantinib and their metabolites with UPLC-MS/MS in rat plasma

As multi-targeted tyrosine kinase inhibitors, sorafenib, regorafenib and cabozantinib are widely used in hepatocellular carcinoma (HCC) for systemic therapies with anti-proliferative and anti-angiogenic effects. Nevertheless, adverse effects or insufficient efficacy appear frequently due to the plasma concentration with individual variability of these drugs. To ensure the curative effect and safety by therapeutic drug monitoring (TDM), this study developed a high throughput method to quantify sorafenib, regorafenib, cabozantinib and their active metabolites in plasma simultaneously. The chromatographic separation analysis achievement was performed on a Waters-ACQUITY UPLC BEH C18 column by UPLC-MS/MS system using a gradient elution of solvent A (acetonitrile) and solvent B (water with 0.1% formic acid) in 3.0 min. This method presented satisfactory results of specificity, precision (the intra-day coefficient of variation was between 2.5% and 6.6%, and the inter-day coefficient of variation was between 4.0% and 11.1%) and accuracy (within ±15% for intra-day and inter-day), as well as the stability under certain conditions, the matrix effect in plasma, and extraction recovery (75.6%–94.4%). The linearity of each analyte in the proper concentration scope indicated excellent. This study strictly complied with the performance rules of assay validation in biological medium proposed by FDA and was successfully applied to the pharmacokinetic study in rats. Thus, it would be an advantageous option to research the relationship between concentration-efficacy and concentration-toxic in HCC patients who were supposed to take these medications.

Determination of Free Amino Acids in Banlangen Granule and its Fractions by Solid Phase Extraction Combined with Ion-pair Hig-h Perfor mance Liquid Chromatography using a Corona-charged Aerosol Detector (SPE-HPLC-CAD)

Background: Banlangen granules are broad-spectrum effective antiviral drugs, and have a large clinical demand in China. Free amino acid is one of the main antiviral active ingredients of Banlangen granules. The pre-processing of samples by the existing pre-column derivatization reversed- HPLC method is complicated. Therefore, the determination of free amino acids (AAs) by underivatized ion-pair HPLC-CAD is advantageous for simplifying the preparation process and improving sensitivity. Objective: To better optimize AAs analysis methods, here a sensitive SPE-HPLC-CAD method with a better resolution was established for the determination of underivatized AAs in Banlangen Granule for the first time. Method: The analytes were separated only by HPLC using a Hypercarb column with gradient elution of solvent A (20 mM nonafluorovaleric Acid in water) and solvent B (0.3% trifluoroacetic acid in acetonitrile-0.3% trifluoroacetic acid in water (1:9, v/v)) at a flow rate of 0.15 mL/min. N2 gas pressure and evaporation temperature of CAD were held at a constant 58.6 psi and 60 ℃, respectively. Results: This method was linear over the respective concentration range of six amino acids. The precision, accuracy, stability and recovery were satisfactory in all samples examined. And the method was successfully applied to determination of free amino acids in Banlangen granules and its fractions. The total contents of six amino acids in 28 batches of Banlangen Granule were between 1.36 mg/g-11.62 mg/g. Conclusion: The proposed method could be a simple, accurate and sensitive alternative approach for the determination of free AAs in Banlangen Granule.

Discovering the major metabolites of the three novel fentanyl analogues 3-methylcrotonylfentanyl, furanylbenzylfentanyl, and 4-fluorocyclopropylbenzylfentanyl for forensic case work

The highly potent opioid analgesic fentanyl and its analogues are involved in an increasing number of overdose deaths worldwide. New fentanyl analogues are continuously emerging, and there is a lack of knowledge concerning the metabolism of these compounds. The determination of fentanyl analogues can be challenging due to their low circulating concentrations and rapid and extensive metabolism, making metabolite identification necessary for confirming drug intake. The aim of this study was to discover and elucidate the structures of the major metabolites of the three novel fentanyl analogues 3-methylcrotonylfentanyl (3-MCF), furanylbenzylfentanyl (FBF), and 4-fluorocyclopropylbenzylfentanyl (4-FCBF), which were all seized at European borders in 2018. 3-MCF, FBF, or 4-FCBF was incubated with human liver microsomes and human hepatocytes for up to 4 h. The metabolites formed were separated by ultra-high-performance liquid chromatography using an octadecyl silica column employing solvent gradient elution with a mobile phase consisting of ammonium formate and methanol. The compounds were detected by quadrupole time-of-flight mass spectrometry. The major metabolites of 3-MCF were formed by N-dealkylation, carboxylation, oxidation, or hydroxylation of the 3-methyl-2-butene, and hydroxylation of both the 3-methyl-2-butene and the piperidine ring. FBF was metabolized through N-dealkylation, amide hydrolysis with/without subsequent hydroxylation at the N-phenyl, and dihydrodiol formation at the furan ring. 4-FCBF metabolism was dominated by N-dealkylation and N-oxidation at the piperidine ring. In the present study, we successfully discovered and elucidated the structures of the major metabolites of 3-MCF, FBF, and 4-FCBF which could be used as markers to confirm intake of these compounds in forensic case work.

A robust RP-HPLC method for determination of turmeric adulteration

A gradient reverse phase high pressure liquid chromatographic method has been proposed for simultaneous determination and separation of curcumin (CUR), bisdemethoxycurcumin (BMC), demethoxycurcumin (DMC), and metanil yellow (MET). The separation was achieved on an Agilent Eclipse XDB-C18 (4.6 mm × 150 mm, 3.5 µm) analytical column using photodiode array (PDA) detection at 425 nm. Gradient elution of solvent A (mixture of 0.1% trifluoro acetic acid and 0.1% formic acid 50:50 v/v) and solvent B (acetonitrile) at a flow rate of 0.8 mL/min was used to accomplish adequate separation of the analytes. The developed method was extensively validated with respect to linearity, detection and quantitation limits, precision, recovery, and robustness testing. Method variables, viz., mobile phase flow rate (0.90 ± 0.05 mL/min), percentage of B at start of gradient (40 ± 2%) and detection wavelength (425 ± 5 nm) were studied by a Box–Behnken Design (BBD) for testing the robustness of the proposed method. The method was found robust, with no significant variations in the method performance, linear in the range of 10–80 µg/mL (r2 = 0.999) and precise (RSD ˂ 2%) satisfying regulatory criteria. The method is selective for rapid determination of turmeric adulteration with a detection limit of 0.37–2.48 µg/mL.

Optimal Conditions for the Direct RP-HPLC Determination of Underivatized Amino Acids with Online Multiple Detection.

The combined use of a dual-UV detector as well as a fluorimetric and a multielectrode electrochemical detector (equipped with a dual electrode, consisting of a conventional size 3mm diameter glassy carbon electrode (GCE) and of a pair of 30μm thick carbon microfibers) is proposed for the detection of the following 15 underivatized amino acids: L-histidine (His), L-cysteine (Cys), creatine (Crn), S-methyl-L-cysteine (Me-Cys), DL-homocysteine (Hcy), L-methionine (Met), beta-(3,4-dihydroxyphenyl)-L-alanine (DOPA), L-tyrosine (Tyr), DL-m-tyrosine (m-Tyr), L-a-methyl-DOPA (Me-DOPA), L-phenylalanine (Phe), DL-alpha-methyltyrosine (Me-Tyr), 5-hydroxy-tryptophan (5-HTP), 3-nitro-L-tyrosine (NO2Tyr), and L-tryptophan (Trp), as well as of 2 dipeptides L-cystathionine (Cysta) and L-carnosine (Car), and of creatinine (Cre). A multilinear solvent (acetonitrile) gradient elution program, determined by a simple optimization algorithm, is required for the efficient reversed-phase separation of the above mixture of 18 solutes within 27min at a flow rate of 1.0mL/min and at 25°C.

Development of a Stability Indicating Method for Simultaneous Analysis of Five Water-Soluble Vitamins by Liquid Chromatography

Abstract Background Water-soluble vitamins are often included simultaneously in pharmaceutical formulations as food complements or in parenteral nutrition mixtures. Given their sensitivity to heat, light or pH variations, it is important to study their stability using validated stability indicating methods. We thus aimed to validate a liquid chromatography (LC) stability-indicating method for the simultaneous quantification of 5 water-soluble vitamins. Methods We analyzed four water-soluble B vitamins (nicotinamide, pyridoxine, folic acid, cyanocobalamin) and ascorbic acid using a LC method with diode array detector. They were separated on a C18 stationary phase under gradient elution of solvent A [0.2 % of metaphosphoric acid in water and acetonitrile 98:2] and solvent B (100 % acetonitrile). All vitamins were subjected to forced degradation conditions and we showed that the obtained degradation products didn’t interfere with the vitamins. Results The method allows the separation of the 5 water-soluble vitamins in a 30 minute run without any interference from the breakdown products obtained with acid/alkaline solutions, hydrogen peroxide, temperature and light. It meets all the qualitative and quantitative criteria for validation with an acceptable accuracy and good linearity. Conclusions This stability-indicating method can be used for carrying out stability studies of water-soluble vitamins in pharmaceutical preparations.

Chemical composition separation of a propylene–ethylene random copolymer by high temperature solvent gradient interaction chromatography

A propylene–ethylene random copolymer was fractionated by preparative temperature rising elution fractionation (TREF). The structural heterogeneity of the bulk sample and its TREF fractions was studied by high temperature liquid chromatography with a solvent gradient elution from 1-decanol to 1,2,4-trichlorobenzene. HPLC alone cannot resolve those propylene-ethylene copolymers with high ethylene content in the bulk sample, due to their low weight fractions in the bulk sample and a small response factor of these components in the ELSD detector, as well as their broad chemical composition distribution. These components can only be detected after being separated and enriched by TREF followed by HPLC analysis. Chemical composition separations were achieved for TREF fractions with average ethylene contents between 2.1 and 22.0mol%, showing that copolymers with higher ethylene contents were adsorbed stronger in the Hypercarb column and eluted later. All TREF fractions, except the 40°C fraction, were relatively homogeneous in both molar mass and chemical composition. The 40°C fraction was rather broad in both molar mass and chemical composition distributions. 2D HPLC showed that the molar masses of the components containing more ethylene units were getting lower for the 40°C fraction. HPLC revealed and confirmed that co-crystallization influences the separation in TREF of the studied propylene-ethylene copolymer.

Development and Application of a Validated UHPLC Method for the Determination of Atropine and Its Major Impurities in Antidote Treatment Nerve Agent Auto-Injectors (ATNAA) Stored in the Strategic National Stockpiles

The development and implementation of advanced analytical technologies is essential for extending the expiry for complex drug products stored in the Strategic National Stockpiles. Consequently, a novel Ultra High-Performance Liquid Chromatographic (UHPLC) method has been developed for the analysis of atropine and its respective impurities to support the analytical research platform for auto-injectors. This study is part of a larger research effort to improve the efficiency and broaden the applicability of advanced analytical methods for medical counter-measure medications. The current HPLC compendial methodology for atropine sulfate injection requires an analysis time of 40 minutes for atropine. In comparison, the novel gradient UHPLC method required only 8 minutes to evaluate both atropine and its major pharmaceutical impurities. Improved separation was achieved on a Waters Acquity UHPLC BEH C18 1.7 μm, 2.1 × 100 mm column employing gradient elution of mobile phase solvent A (0.1% H3PO4) and solvent B (0.1% H3PO4, 90% ACN, and 10% H2O). The method was validated according to USP Category I requirements for assay. The daily standard calibration curves were linear over a concentration range from 50 μg/mL to 250 μg/mL with a correlation coefficient of >0.999. The detection limit (LOD) and quantitation limit (LOQ) were 3.9 μg/ml and 13.1 μg/ml, respectively. Resolution results indicate that atropine and the following impurities, degradants and a preservative can also be separated and analyzed using this proposed method: noratropine, 4,4’-di-hy-droxydiphenyl ether, 2,4’-dihydroxydiphenyl ether, 4-bromophenol, 4-hydro-xyatropine, tropic acid, apoatropine HCl, atropic acid, hydroquinone, nitroethane, phenol and catechol. The UHPLC method demonstrated enhanced selectivity and significantly reduced the analysis time when compared with the traditional USP compendial HPLC method. The method was successfully applied to the evaluation of atropine in ATNAA auto-injectors lots from the Strategic National Stockpiles.

Temperature-assisted solute focusing with sequential trap/release zones in isocratic and gradient capillary liquid chromatography: Simulation and experiment

In this work we characterize the development of a method to enhance temperature-assisted on-column solute focusing (TASF) called two-stage TASF. A new instrument was built to implement two-stage TASF consisting of a linear array of three independent, electronically controlled Peltier devices (thermoelectric coolers, TECs). Samples are loaded onto the chromatographic column with the first two TECs, TEC A and TEC B, cold. In the two-stage TASF approach TECs A and B are cooled during injection. TEC A is heated following sample loading. At some time following TEC A’s temperature rise, TEC B’s temperature is increased from the focusing temperature to a temperature matching that of TEC A. Injection bands are focused twice on-column, first on the initial TEC, e.g. single-stage TASF, then refocused on the second, cold TEC. Our goal is to understand the two-stage TASF approach in detail. We have developed a simple yet powerful digital simulation procedure to model the effect of changing temperature in the two focusing zones on retention, band shape and band spreading. The simulation can predict experimental chromatograms resulting from spatial and temporal temperature programs in combination with isocratic and solvent gradient elution. To assess the two-stage TASF method and the accuracy of the simulation well characterized solutes are needed. Thus, retention factors were measured at six temperatures (25–75°C) at each of twelve mobile phases compositions (0.05-0.60 acetonitrile/water) for homologs of n-alkyl hydroxylbenzoate esters and n-alkyl p-hydroxyphenones. Simulations accurately reflect experimental results in showing that the two-stage approach improves separation quality. For example, two-stage TASF increased sensitivity for a low retention solute by a factor of 2.2 relative to single-stage TASF and 8.8 relative to isothermal conditions using isocratic elution. Gradient elution results for two-stage TASF were more encouraging. Application of two-stage TASF increased peak height for the least retained solute in the test mixture by a factor of 3.2 relative to single-stage TASF and 22.3 compared to isothermal conditions for an injection four-times the column volume. TASF improved resolution and increased peak capacity; for a 12-min separation peak capacity increased from 75 under isothermal conditions to 146 using single-stage TASF, and 185 for two-stage TASF.

Development and validation of a simple UHPLC–MS/MS method for the simultaneous determination of trimethylamine N-oxide, choline, and betaine in human plasma and urine

A simple, sensitive, and precise ultra-high performance liquid chromatography–tandem mass spectrometry method was developed and validated for the simultaneous determination of trimethylamine N-oxide, choline, and betaine in human plasma and urine. Sample preparation involved protein precipitation with methanol containing internal standards. Chromatographic separation was achieved using an Acquity BEH Amide (2.1mm×50mm, 1.7μm) analytical column with gradient elution of solvent A (10mM ammonium formate, pH 3.5) and solvent B (acetonitrile). The flow rate was 0.4mL/min and the total run time was 5min. Detection of analytes was performed using heated electrospray ionization (positive mode) and selected reaction monitoring. Excellent linearity was observed over the standard curve concentration ranges of 0.010–5.00μg/mL (plasma) and 1.00–150μg/mL (urine) for all analytes. The intra- and inter-day accuracy and precision for all quality controls were within ±10%. Excellent recovery was observed. The method is rapid, accurate and reproducible, and was successfully applied to a pilot study of markers of atherosclerosis in patients with kidney disease who underwent successful kidney transplantation.

Dual gradient macroporous resin column chromatography for concurrent separation and purification of three families of marine bacterial lipopeptides from cell free broth

A simple and efficient dual-gradient elution strategy for the separation and purification of marine bacterial lipopeptides was developed in macroporous resin column chromatography. Preliminary batch adsorption studies conducted on four different resins revealed that a non-polar resin, HP-20, exhibited the highest adsorption capacity. The optimal conditions yielding maximum adsorption of lipopeptides on to this resin were as follows: temperature=40°C, resin dosage=10gL−1 and initial lipopeptide concentration=3gL−1. While employing a simple stepwise solvent gradient elution under optimal conditions, three lipopeptide families, namely, iturin, fengycin and surfactin could be separated with poor resolution. However, the resolution was significantly improved after implementing a family specific dual-gradient elution strategy, wherein, both the solvent composition and pH of the mobile phase were simultaneously varied. Using this approach, lipopeptides were effectively resolved and purities up to 68.3% (iturin), 77.6% (fengycin) and 91.6% (surfactin) were achieved. To the best of our knowledge, this is the first report on the macroporous adsorption resin (MAR) chromatography employing family specific dual-gradient elution strategy for simultaneous recovery and purification of lipopeptides directly from cell free culture broth.

Simultaneous determination of chlorogenic acid, caffeic acid, alantolactone and isoalantolactone in Inula helenium by HPLC.

A rapid and sensitive high-performance liquid chromatographic (HPLC) method was developed for the simultaneous separation and determination of chlorogenic acid, caffeic acid, alantolactone and isoalantolactone in Inula helenium. The HPLC separation was performed on an Elite Hypersil C18 column (200 × 4.6 mm i.d., 5 µm particle size) with a gradient elution of solvent A (acetonitrile) and solvent B (0.1% phosphoric acid in water) at a flow rate of 1.0 mL/min. Detection was monitored at 225 nm. The recovery of chlorogenic acid ranged from 95.6 to 107.7%, the recovery of caffeic acid ranged from 95.4 to 104.2%, the recovery of alantolactone ranged from 95.8 to 100.8% and the recovery of isoalantolactone ranged from 96.5 to 102.3%. The retention times for chlorogenic acid, caffeic acid, alantolactone and isoalantolactone were 5.2, 7.1, 25.6 and 26.6 min with the limits of detection of 0.069, 0.021, 0.039 and 0.051 µg/mL, respectively. Relative standard deviation for the intra-day and inter-day was ≤2.5%. The validated method is reliable for the routine control of these four compounds in I. helenium.

QUANTITATIVE SIMULTANEOUS DETERMINATION OF FENOFIBRATE, ATORVASTATIN, AND EZETIMIBE IN TABLETS USING GRADIENT HIGH-PERFORMANCE COLUMN LIQUID CHROMATOGRAPHY AND HIGH-PERFORMANCE THIN-LAYER CHROMATOGRAPHY

Simple, accurate, precise, sensitive, and validated high-performance liquid chromatography (HPLC) and high-performance thin-layer chromatography (HPTLC)-densitometric methods were developed for simultaneous determination of fenofibrate (FEN), atorvastatin (ATO), and ezetimibe (EZE) in combined tablet dosage form. In Method A, the gradient RP-HPLC analysis was performed on a Shim-pack C18 column (150 × 6 mm id), using a mobile phase consisting of 0.1% formic acid and acetonitrile in solvent gradient elution for 25 min at a flow rate of 1.5 mL min−1. Quantification was carried out using a photodiode array UV detector at 245 nm. The employment of diode array detector allowed selectivity confirmation by peak purity evaluation. In Method B, the HPTLC analysis was carried out on an aluminum-backed sheet of silica gel 60F254 layer using toluene: methanol:triethylamine (8:1.5:0.1, v/v/v) as the mobile phase. Quantification was achieved with UV densitometry at 245 nm. The analytical methods were validated according to International Conference on Harmonization guidelines. Low relative standard deviation values indicated good precision. Both the methods were successfully applied for the analysis of drugs in laboratory-prepared mixtures and commercial tablets. No chromatographic interference from tablet excipients was found, and hence these methods are applicable for simultaneous determination of FEN, ATO, and EZE in pharmaceutical formulations.

Simultaneous Quantification of Sphingolipids in Small Quantities of Liver by LC-MS/MS.

Sph, S1P, and Cer, derived from the membrane sphingolipids, act as intracellular and intercellular mediators, involved in various (path) physiological functions. Accordingly, determining the distributions and concentrations of these sphingolipid mediators in body tissues is an important task. Consequently, a method for determination of sphingolipids in small quantities of tissue is required. Sphingolipids analysis has been dependent on improvements in mass spectrometry (MS) technology. Additionally, decomposition of sphingosine-1-phosphate (S1P) in the tissue samples before preparation for MS has hindered analysis. In the present study, a method for stabilization of liver samples before MS preparation was developed using a heat stabilizer (Stabilizor™ T1). Then, a LC-MS/MS method using a triple-quadrupole mass spectrometer with a C8 column was developed for simultaneous determination of sphingolipids in small quantities of liver specimens. This method showed good separation and validation results. Separation was performed with a gradient elution of solvent A (5 mmol L(-1) ammonium formate in water, pH 4.0) and solvent B (5 mmol L(-1) ammonium formate in 95% acetonitrile, pH 4.0) at 300 μL min(-1). The lower limit of quantification was less than 132 pmol L(-1), and this method was accurate (∼13.5%) and precise (∼7.13%) for S1P analysis. The method can be used to show the tissue distribution of sphingolipids.

Pharmacokinetics and tissue distribution study of tanshinone IIA after oral administration of Bushen Huoxue Qubi granules to rats with blood stasis syndrome.

Background:Bushen Huoxue Qubi (BHQ) granules, a traditional Chinese medicine preparation, has been clinically used for the treatment of the blood stasis syndrome.Objective:The main objective is to investigate whether the diseased condition would alter the pharmacokinetics and tissue distribution of tanshinone IIA in BHQ, which was given orally to the acute blood stasis rats.Materials and Methods:The main bioactive constituent in BHQ, tanshinone IIA, was measured in the plasma and tissues of animals by the high performance liquid chromatography with ultraviolet detection. The analysis was successfully performed on an Agilent TC-C18 column (250 × 4.6 mm I.D., 5 μm) protected with a Octadecylsilane (ODS) guard column (10 × 4.6 mm I.D., 5 μm). The mobile phase was aqueous solution (A) (containing 0.40% aqueous acetic acid) and acetonitrile (B). The conditions of the solvent gradient elution were 35-40% (B) in 0-15 min, 40-42% (B) in 15-18 min and 42-70% (B) in 18-30 min at a flow rate of 1.0 mL/min. Detection was conducted with wavelength of 270 nm at 30°C.Results:Good linearity relationships were found (r2> 0.9955) over the investigated concentration range for bio-samples. Blood stasis was associated with significantly higher area under the concentration-time curve (AUC), the maximum plasma concentration (Cmax) and biological half-life (t1/2), lower total body clearance (CL) and apparent volume of distribution (Vd) of tanshinone IIA in plasma and higher AUC0-t of tanshinone IIA in the analyzed tissues of rats treated with BHQ.Conclusion:Blood stasis could alter pharmacokinetics and tissue distribution of tanshinone IIA in BHQ.

Determination of warfarin alcohols by ultra-high performance liquid chromatography–tandem mass spectrometry: Application to in vitro enzyme kinetic studies

A sensitive, accurate, and reproducible ultra-high performance liquid chromatography-tandem mass spectrometry method was developed and validated for determination of warfarin and its alcohol metabolites (RS/SR- and RR/SS-warfarin alcohol) in 10mM Tris-HCl incubation buffer (pH 7.4). Sample preparation involved acidification with 4% formic acid, followed by liquid-liquid extraction using methyl tert-butyl ether. Chromatographic separation was achieved using a Hypersil Gold C18 (2.1mm×100mm, 1.9μm) analytical column with gradient elution of solvent A (water containing 0.01% formic acid) and solvent B (acetonitrile containing 0.1% formic acid). The flow rate was 0.4mL/min and the total run time was 5min. Detection of analytes was performed using heated electrospray ionization (negative mode) and selected reaction monitoring. Excellent linearity was observed for all analytes over the standard curve concentration ranges of 100-10,000ng/mL for warfarin, and 0.5-250ng/mL for warfarin alcohols. The intra- and inter-day accuracy and precision for analytes were within ±10.0%. Excellent recovery and negligible matrix effects were observed. The method is robust, sensitive, accurate and reproducible, and was successfully applied to in vitro enzyme kinetic studies of warfarin.

Improved chemical composition separation of ethylene–propylene random copolymers by high-temperature solvent gradient interaction chromatography

High-temperature solvent gradient interaction chromatography (HT-SGIC) is a fast and efficient fractionation technique for the chemical composition analysis of olefin copolymers. The separation of ethylene-propylene random copolymers (EPRs) was achieved on a graphitic stationary phase, Hypercarb, at 160 °C by using linear solvent gradient elution from 1-decanol to 1,2,4-trichlorobenzene (TCB). In the present work, the solvent gradient profile was modified to improve the chromatographic separation of EPRs. With the aim to obtain a better resolution in separation, a slow increase in the volume fraction of TCB was applied. This allowed for a relatively large retention region for linear polyethylene (PE) chains on the column; thereby, a broader elution volume zone between the start of the gradient and the PE elution was achieved. The efficiency of this new gradient profile was demonstrated by analysing two fully amorphous EPR samples. Clear differences in the chemical composition of these EPR samples with similar ethylene contents have been proven by using this modified solvent gradient. The comprehensive chemical composition and microstructure analysis of the SGIC-separated fractions by FTIR revealed that ethylene/propylene (EP) copolymer chains were eluted according to their ethylene/propylene contents and E or P sequence lengths, even though they are distributed in a random manner. These results showed that the solvent composition is an important factor to affect the interactive adsorption or desorption behaviour of EP chains on Hypercarb. In this way, for the first time, the determination of the complex composition and chain structure of EPR samples was achieved within short analysis time, which is not possible till now using other fractionation techniques reported.

QUANTITATIVE SIMULTANEOUS DETERMINATION OF AMLODIPINE, VALSARTAN, AND HYDROCHLOROTHIAZIDE IN “EXFORGE HCT” TABLETS USING HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY AND HIGH-PERFORMANCE THIN-LAYER CHROMATOGRAPHY

Simple, accurate, precise, sensitive, and validated HPLC and HPTLC-densitometric methods were developed for simultaneous determination of amlodipine (AML), valsartan (VAL), and hydrochlorothiazide (HYD) in combined tablet dosage form. Method A, the gradient RP-HPLC analysis was performed on a Phenomenex Luna C18 (4.60 mm × 150 mm, 5 µ particle size) column, using a mobile phase consisting of 10 mM ammonium acetate buffer (pH 6.7) and methanol in solvent gradient elution for 20 min at a flow rate of 1 mL min−1. Quantification was carried out using a photodiode array UV detector at 238 nm. The employment of a diode array detector allowed selectivity confirmation by peak purity evaluation. Method B, the HPTLC analysis was carried out on an aluminum-backed sheet of silica gel 60F254 layers using chloroform: glacial acetic acid:n-butyl acetate (8:4:2, v/v/v) as the mobile phase. Quantification was achieved with UV densitometry at 320 nm. The analytical methods were validated according to International Conference on Harmonization (ICH) guidelines. No chromatographic interference from tablet excipients was found and hence these methods are applicable for simultaneous determination of AML, VAL, and HYD in pharmaceutical formulations and biological fluids.