Reversed-phase high-performance liquid chromatography method for simultaneous analysis of two liposome-formulated short interfering RNA duplexes

Gene silencing induced by short interfering RNA (siRNA) has proven to be useful in genomic research and has great potential for therapeutic applications; however, siRNAs are not readily bioavailable. Cationic liposomes offer effective protection of drug product from nucleases and enable distribution to desired target organs. The amount of siRNA in the formulation must be determined accurately. We have developed a stability-indicating, ion-pair, reversed-phase high-performance liquid chromatography method to separate and accurately quantitate two siRNA duplexes in a liposome without sample pretreatment. The gradient mobile phase system consisted of 385 mM hexafluoro-2-propanol, 14.5 mM triethylamine, and 5% methanol (mobile phase A) and 385 mM hexafluoro-2-propanol, 14.5 mM triethylamine, and 90% methanol (mobile phase B). The column used was an XBridge C18 column (50 × 2.1 mm i.d., 2.5 μm particle size), and separation was performed at 60 °C. Quantitation was achieved with ultraviolet (UV) detection at 260 nm. Linearity was established for the single strands of both siRNA duplexes for concentrations ranging from 10 to 110 μg/ml. Accuracy of the method was determined by replicate analysis (n = 5) at four concentrations (R² > 0.996 and relative standard deviations [RSDs] of 1-4%). The use of an ion-pairing reagent that is compatible with mass spectrometry detection makes this method amenable to liquid chromatography-mass spectrometry (LC-MS) impurity profiling.     

Simultaneous determination of aminomethylbenzoic acid,cefminox sodium and etamsylate in human urine by capillary electrophoresis

A sensitive and selective capillary electrophoresis method is developed, for the first time, for effective separation and simultaneous determination of aminomethylbezoic acid (PAMBA), cefminox sodium (CMNX) and etamsylate (ETM). The electrophoresis conditions were investigated and optimized. A 25 mM phosphate solution (pH 8.5) was used as a buffer and the peak area was determined with UV detection at 216 nm wavelength under 18 kV separation voltage. Under optimal conditions, the three drugs can be separated effectively. Good linearity was achieved in 3.13–150 μg/mL for PAMBA, 6.25–150 μg/mL for CMNX and 3.13–150 μg/mL for ETM, with the correlation coefficients of >0.999. The limit of detection (LOD) for PAMBA, CMNX and ETM was 1.04, 2.08 and 1.04 μg/mL, respectively. Their recoveries in human urine were in the range from 90.2% to 101% with the RSD (n = 5) of 0.7–3.1%. The proposed method is simple, rapid and accurate, and provides the sensitivity and linearity necessary for analysis of the test drugs in human urine at clinically relevant concentrations.     

Chemiluminescent immunoassay of thyroxine enhanced by microchip electrophoresis

A homogeneous chemiluminescent immunoassay of thyroxine (T4) enhanced by microchip electrophoresis separation has been developed. The method deployed the competitive immunoreaction of T4 and horseradish peroxidase (HRP)-labeled T4 (HRP-T4) with anti-T4 mouse monoclonal antibody (Ab). HRP-T4 and the HRP-T4-Ab complex were separated and quantified by using microchip electrophoresis (MCE) with chemiluminescence (CL) detection. Highly sensitive CL detection was achieved by means of HPR-catalyzed luminol-H₂O₂ reaction. Due to the effective MCE separation, the CL analytical signal was less prone to sample matrix interference. Under the selected assay conditions, the MCE separation was accomplished within 60 s. The linear range for T4 was 5-250 nM with a detection limit of 2.2 nM (signal/noise ratio = 3). The current method was successfully applied for the quantification of T4 in human serum samples. It was demonstrated that the current MCE-CL-enhanced competitive immunoassay was quick, sensitive, and highly selective. It may serve as a tool for clinical analysis of T4 to assist in the diagnosis of thyroid gland functions.     

A capillary electrophoresis-based enzyme assay for kinetics and inhibition studies of carbonic anhydrase

In the current study, capillary electrophoresis (CE)-based enzyme assay for characterization and inhibition study of bovine carbonic anhydrase II (bCA II) was developed. The developed method is the first CE assay for carbonic anhydrase (CA). The method was optimized in order to get short analysis time, minimal sample volume consumption, and high resolution of substrate and product. The CE conditions were optimized as follows: fused-silica capillary (30 cm effective length × 75 μm i.d.), pressure injection for 5 s, 20 mM sodium borate buffer (pH 9.0), constant voltage of 15 kV, constant capillary temperature of 25 °C, and detection at 260 nm. For precise measurements, uridine was used as an internal standard during optimization of the CE methods. The limits of detection and quantification for p-nitrophenyl acetate (p-NPA) were 3.01 and 9.12 μM, respectively, whereas for p-nitrophenolate they were 2.05 and 6.22 μM, respectively. The performance of the developed method was confirmed by determination of kinetic parameters (i.e., K_m and V_max of bCA for p-NPA); the inhibition constant (K_i) was determined for furosemide, a standard inhibitor of CA. The new method proved to be fast and efficient, and it can be used for the investigation of inhibitors of all isoforms of CAs.     

Determination of the specific activities of methionine sulfoxide reductase A and B by capillary electrophoresis

A capillary electrophoresis (CE) method for the determination of methionine sulfoxide reductase A and methionine sulfoxide reductase B activities in mouse liver is described. The method is based on detection of the 4-(dimethylamino)azobenzene-4′-sulfonyl derivative of l-methionine (dabsyl Met), the product of the enzymatic reactions when either dabsyl l-methionine S-sulfoxide or dabsyl l-methionine R-sulfoxide is used as a substrate. The method provides baseline resolution of the substrates and, therefore, can be used to easily determine the purity of the substrates. The method is rapid (∼20 min sample to sample), requires no column regeneration, and uses very small amounts of buffers. Separation was performed by using a 75-μm internal diameter polyimide-coated fused silica capillary (no inside coating) with 60 cm total length (50 cm to the detector window). Samples were separated at 22.5 kV, and the separation buffer was 25 mM KH₂PO₄ (pH 8.0) containing 0.9 ml of N-lauroylsarcosine (sodium salt, 30% [w/v] solution) per 100 ml of buffer. Prior to use, the capillary was conditioned with the same buffer that also contained 25 mM sodium dodecyl sulfate. The CE method is compared with high-performance liquid chromatography (HPLC) as determined by comparing results from measurements of hepatic enzyme activities in mice fed either deficient or adequate selenium.     

In vitro ruminal methanogenesis of a hay-rich substrate in response to different combination supplements of nitrocompounds; pyromellitic diimide and 2-bromoethanesulphonate

Sixteen combinations of 5 treatments at 4 levels were designed in a L₁₆(4⁵) orthogonal experimental design to evaluate associative effects of five methanogenesis inhibitors at four dose levels: nitroethane (NE, 0 mM, 5 mM, 10 mM and 15 mM), 2-nitroethanol (NEOH, 0 mM, 5 mM, 10 mM and 15 mM), 2-nitro-1-propanol (NPOH, 0 mM, 5 mM, 10 mM and 15 mM), pyromellitic diimide (PMDI, 0 mM, 0.02 mM, 0.05 mM and 0.07 mM) and 2-bromoethanesulphonate (BES, 0 mM, 0.01 mM, 0.03 mM and 0.05 mM) on in vitro ruminal methane production of the mixed substrate (Chinese wildrye hay:maize meal = 4:1) using a cumulative gas production technique. After 48 h incubation, in vitro dry matter disappearance (IVDMD), total gas production (GP₄₈, ml/g DM) and total volatile fatty acids (VFA) production in various combinations of these inhibitors were decreased by 10.6-56.0, 26.5-44.5 and 20.3-47.6%, respectively (P<0.05). The molar proportion of acetate in the inhibitor combination groups was decreased by 6.6-12.5% while those of propionate and butyrate were increased by 7.0-19.2 and 21.9-56.5% (P<0.01), respectively. Methane proportion (MP) in total gas production was reduced by 79.4-98.5% (P<0.01), and the highest inhibition occurred in the combination of 10 mM NE, 10 mM NPOH, 0.07 mM PMDI and 0.01 mM BES in cultures. The partial correlation coefficients between NE, NEOH, NPOH, PMDI or BES and CH₄ proportion were −0.465 (P<0.01), −0.417 (P<0.01), −0.355 (P<0.05), −0.408 (P<0.01) and −0.345 (P<0.05), respectively, indicating that NE was the most potent inhibitor, followed by NEOH and PMDI, and finally NPOH and BES. In general, VFA production in the inhibitor combinations was substantially shifted to produce much more butyrate and propionate and less acetate. The combination of 15 mM NE, 10 mM NEOH, 5 mM NPOH, 0.07 mM PMDI and 0.01 mM BES in cultures, leading to >95% methane inhibition, may be the optimal application of these inhibitors with less depression of total VFA production. Further feeding trials to validate these combinations is still required on rumen function, methane production, growth performance and milk production.     

Determination of p-aminohippuric acid in rat plasma by liquid chromatography-tandem mass spectrometry

A rapid, simple and sensitive method was developed for the determination of para-aminohippuric acid (PAH) in rat plasma using liquid chromatography tandem mass spectrometry (LC-MS-MS). Acetaminophen was used as the internal standard. Chromatographic separation was performed using a Symmetry C₁₈ column and the mobile phase was composed of A: 2 mM ammonium formate and 0.1% formic acid in water and B: 2 mM ammonium formate and 0.1% formic acid in acetonitrile (ACN) (A:B, 30:70, v/v). Detection was performed on a triple–quadrupole tandem mass spectrometer using positive ion mode electrospray ionization (ESI) in the multiple reaction monitoring (MRM) mode. The MS/MS ion transitions monitored were m/z 195.2 → 120.2 and 152.1 → 110.1 for PAH and acetaminophen, respectively. Good linearity is observed over the concentration range of 0.1–500 μg/ml. The method was proved to be accurate and reliable and was applied to a pharmacokinetic study in rat.     

A quantitative method to assess muscle tissue oxygenation in vivo by monitoring H nuclear magnetic resonance myoglobin resonances

A nuclear magnetic resonance (NMR) method was implemented to assess in vivo oxygenation levels by a quantitative determination of the ¹H NMR myoglobin (Mb) resonances. The proximal His-F8 N_δH at 70-90 ppm and Val-E11 γCH₃ resonance at -2.8 ppm, reflecting deoxygenated (deoxy-Mb) and oxygenated (met-Mb) states, were alternately recorded. The method was developed in vitro choosing a couple of NMR sequences that could each maximize the signal-to-noise ratio (SNR) while avoiding baseline rolling and suppressing the water signal. Two quantitative calibration methods were implemented for deoxy- and met-Mb samples (0.1-1 mM), respectively. The respective limit of detection (LOD) and limit of quantification (LOQ) were 0.015 and 0.05 mM for met-Mb and 0.013 and 0.042 mM for deoxy-Mb. Sequences and calibration curves were employed in vivo in Arenicola marina to obtain, for the first time, an accurate measurement of oxy- and deoxy-Mb actual concentrations. In Arenicola, the peaks at approximately 87 and -2.7 ppm, reflecting the deoxy- and oxy-Mb states, respectively, were alternately recorded during increasing hypoxia. The deoxy-Mb concentrations were obtained from the calibration curve. The oxy-Mb concentrations were calculated from the calibration of met-Mb because it was proved that oxy- and met-Mb gave the same NMR molar response. From oxy- and deoxy-Mb concentrations, the intracellular oxygen partial pressure (P_iO₂) trend was determined.     

A pulsed amperometric detection method of galactose 1-phosphate for galactosemia diagnosis

Galactose 1-phosphate uridyltransferase deficiency causes the accumulation of galactose and galactose 1-phosphate (Gal 1-P) in the blood. We describe a new pulsed amperometric detection method for determining Gal 1-P levels as a pathognomic marker for the diagnosis of galactosemia. The method uses high-performance anion-exchange chromatography with pulsed amperometric detection. In an anion-exchange column, the analytes were separated in 5 min by the eluent mixture of 40 mM NaOH and 40 mM Na₂CO₃. The detection limit (signal to noise ratio of 3) to Gal 1-P was 30 μg/dL. The linear dynamic range was 3.0-50 mg/dL (r² = 0.9999). The mean recoveries of Gal 1-P for intra- and interday assays were 97.55-103.78%. This method clearly separated the type I galactosemia patients from the normal group and is a practical procedure for the rapid diagnosis of galactosemia.     

A simple and sensitive HPLC fluorescence method for determination of tadalafil in mouse plasma

A simple and sensitive high-performance liquid chromatographic (HPLC) method utilizing fluorescence detection was developed for the determination of the phosphodiesterase type 5 inhibitor tadalafil in mouse plasma. This method utilizes a simple sample preparation (protein precipitation) with high recovery of tadalafil (∼98%), which eliminates the need for an internal standard. For constituent separation, the method utilized a monolithic C₁₈ column and a flow rate of 1.0 mL/min with a mobile phase gradient consisting of aqueous trifluoroacetic acid (0.1% TFA in deionized water pH 2.2, v/v) and acetonitrile. The method calibration was linear for tadalafil in mouse plasma from 100 to 2000 ng/mL (r > 0.999) with a detection limit of approximately 40 ng/mL. Component fluorescence detection was achieved using an excitation wavelength of 275 nm with monitoring of the emission wavelength at 335 nm. The intra-day and inter-day precision (relative standard deviation, RSD) values for tadalafil in mouse plasma were less than 14%, and the accuracy (percent error) was within −14% of the nominal concentration. The method was utilized on mouse plasma samples from research evaluating the potential cardioprotective effects of tadalafil on mouse heart tissue exposed to doxorubicin, a chemotherapeutic drug with reported cardiotoxic effects.     

Development and validation of a rapid and sensitive assay for simultaneous quantification of midazolam, 1′-hydroxymidazolam,and 4-hydroxymidazolam by liquid chromatography coupled to tandem mass-spectrometry

Midazolam is an ultra short acting benzodiazepine derivative and a specific probe for phenotyping cytochrome P450 (P450) 3A4/5 activity. A rapid, sensitive, and selective LC–MS/MS method was developed for simultaneous quantitation of midazolam and its metabolites (1′-hydroxymidazolam and 4-hydroxymidazolam). Deuterated (D₅) analog of midazolam was utilized as an internal standard. Sample preparation either from human plasma (100 μL) or liver microsomal incubations involved a simple protein precipitation using acetonitrile (900 μL) with an average recovery of >90% for all compounds. The chromatographic separation was achieved using Zorbax-SB Phenyl, Rapid Resolution HT (2.1 mm × 100 mm, 3.5 μm) and a gradient elution with 10 mM ammonium acetate in 10% methanol (A) and acetonitrile (B). The flow rate was 0.25 mL/min and total run time was 5.5 min. Calibration curves were linear over the concentration range of 0.100–250 ng/mL. The lower limit of quantitation (LLOQ) was 0.1 ng/mL for all three analytes. The accuracy and precision, estimated at LLOQ and three concentration levels of quality control samples in six replicates, were within 85–115%. In conclusion, a robust, simple and highly sensitive analytical method was developed and validated for the analysis of midazolam and its metabolites. This method is suitable for characterizing the P450 3A4/5 activity in vitro or in human pharmacokinetic studies allowing administration of smaller doses of midazolam.     

Novel assays for proteases using green fluorescent protein-tagged substrate immobilized on a membrane disk

Green fluorescent protein (GFP) is very stable for various proteases. Using this property, three protease assay methods designated the disk separation assay for remaining GFP (DSAR), the disk separation assay for liberated GFP (DSAL), and the homogeneous assay for fluorescence concentrated on membrane (HAFCOM) were developed. These methods employ a nylon membrane designated “Cleave-Checker” on which GFP-SpA_B (domain B in staphylococcal protein A) is immobilized. The SpA_B region was used as a substrate for the protease, and the isolation of GFP from the membrane generated by the digestion of the SpA_B region was detected. In DSAR, it was possible to detect solution of at least 25 ng/ml trypsin or proteinase K by visual observation. The most important feature of DSAR is that the detection of the protease is possible only under UV light. In contrast, DSAL is suitable for a highly sensitive assay. The assay ranges of DSAL were 1.6 to 100 ng/ml in trypsin and 1.6 to 400 ng/ml in proteinase K. HAFCOM does not require bound/free (B/F) separation; thus, the procedure is simpler than that with DSAL and the reproducibility is high. The assay ranges of HAFCOM were 25 to 400 ng/ml in trypsin and 12.5 to 200 ng/ml in proteinase K. The Cleave-Checker used for these methods was stable in a dry state, and long-term preservation for at least several months was possible.     

Combined use of [TBA][L-ASP] and hydroxypropyl-β-cyclodextrin as selectors for separation of Cinchona alkaloids by capillary electrophoresis

In this paper, a new capillary electrophoresis (CE) separation and detection method was developed for the chiral separation of the four major Cinchona alkaloids (quinine/quinidine and cinchonine/cinchonidine) using hydroxypropyl-β-cyclodextrin (HP-β-CD) and chiral ionic liquid ([TBA][L-ASP]) as selectors. Separation parameters such as buffer concentrations, pH, HP-β-CD and chiral ionic liquid concentrations, capillary temperature, and separation voltage were investigated. After optimization of separation conditions, baseline separation of the three analytes (cinchonidine, quinine, cinchonine) was achieved in fewer than 7 min in ammonium acetate background electrolyte (pH 5.0) with the addition of HP-β-CD in a concentration of 40 mM and [TBA][L-ASP] of 14 mM, while the baseline separation of cinchonine and quinidine was not obtained. Therefore, the first-order derivative electropherogram was applied for resolving overlapping peaks. Regression equations revealed a good linear relationship between peak areas in first-order derivative electropherograms and concentrations of the two diastereomer pairs. The results not only indicated that the first-order derivative electropherogram was effective in determination of a low content component and of those not fully separated from adjacent ones, but also showed that the ionic liquid appeared to be a very promising chiral selector in CE.     

ZnO–CuxO/polypyrrole nanocomposite modified electrode for simultaneous determination of ascorbic acid,dopamine, and uric acid

Novel zinc oxide (ZnO) nanosheets and copper oxide (Cu_xO, CuO, and Cu₂O) decorated polypyrrole (PPy) nanofibers (ZnO–Cu_xO–PPy) have been successfully fabricated for the simultaneous determination of ascorbic acid (AA), dopamine (DA), and uric acid (UA). The morphology and structure of ZnO–Cu_xO–PPy nanocomposites were characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), and Raman spectroscopy. Compared with the bare glassy carbon electrode (GCE), PPy/GCE, Cu_xO–PPy/GCE, and ZnO–PPy/GCE, ZnO–Cu_xO–PPy/GCE exhibits much higher electrocatalytic activities toward the oxidation of AA, DA, and UA with increasing peak currents and decreasing oxidation overpotentials. Cyclic voltammetry (CV) results show that AA, DA, and UA could be detected selectively and sensitively at ZnO–Cu_xO–PPy/GCE with peak-to-peak separation of 150 and 154 mV for AA–DA and DA–UA, respectively. The calibration curves for AA, DA, and UA were obtained in the ranges of 0.2 to 1.0 mM, 0.1 to 130.0 μM, and 0.5 to 70.0 μM, respectively. The lowest detection limits (signal/noise = 3) were 25.0, 0.04, and 0.2 μM for AA, DA, and UA, respectively. With good selectivity and sensitivity, the current method was applied to the determination of DA in injectable medicine and UA in urine samples.     

Liquid chromatography–tandem mass spectrometry quantification of levosulpiride in human plasma and its application to bioequivalence study

An improved method for determining levels of levosulpiride in human plasma using ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) was developed and validated. The protein precipitation method was used for plasma sample preparation. Levosulpiride and an internal standard (IS) were isocratically separated on a UPLC BEH C₁₈ column with a mobile phase of ammonium formate buffer (1 mM, adjusted to pH 3 with formic acid) and acetonitrile (60:40, v/v). MS/MS detection was performed by monitoring the parent → daughter pair of levosulpiride and the IS at m/z 342 → 112 and 329 → 256, respectively. The method was linear from 2.5 to 200 ng/mL and exhibited acceptable precision and percent recovery. The method was successfully demonstrated in pharmacokinetic and bioequivalence studies of two levosulpiride oral formulations administered to healthy volunteers. When compared to the previous LC–MS methods, the proposed method is faster, well-validated, and uses lesser plasma volume and a similar sensitivity. The use of UPLC allowed rapid and sensitive quantification of levosulpiride, making this method suitable for high-throughput clinical applications.     

Development of an enzymatic chromatography strip with nicotinamide adenine dinucleotide–tetrazolium coupling reactions for quantitative l-lactate analysis

In this study, a dry assay of l-lactate via the enzymatic chromatographic test (ECT) was developed. An l-lactate dehydrogenase plus a nicotinamide adenine dinucleotide (NADH) regeneration reaction were applied simultaneously. Various tetrazolium salts were screened to reveal visible color intensities capable of determining the lactate concentrations in the sample. The optimal analysis conditions were as follows. The diaphorase (0.5 μl, 2⁻⁶ U/μl) was immobilized in the test line of the ECT strip. Nitrotetrazolium blue chloride (5 μl, 12 mM), l-lactate dehydrogenase (1 μl, 0.25 U/μl), and NAD⁺ (2 μl, 1.5 × 10⁻⁵ M) were added into the mobile phase (100 μl) composed of 0.1% (w/w) Tween 20 in 10 mM phosphate buffer (pH 9.0), and the process was left to run for 10 min. This detection had a linear range of 0.039 to 5 mM with a detection limit of 0.047 mM. This quantitative analysis process for l-lactate was easy to operate with good stability and was proper for the point-of-care testing applications.     

An optimized and validated RP-HPLC/UV detection method for simultaneous determination of all-trans-Retinol (Vitamin A) and α-Tocopherol (Vitamin E) in human serum: Comparison of different particulate reversed-phase HPLC columns

A novel, simple and fast reversed-phase HPLC/UV method was developed, optimized for various chromatographic conditions, and validated according to international guidelines for simultaneous determination of all-trans-retinol and α-tocopherol in human serum using retinyl acetate as internal standard in the concentration of 0.5 μg/ml. A liquid-phase extraction was applied to the 250 μl of serum with n-hexane–dichloromethane mixture (70:30, v/v), in two steps, using ethanol–methanol mixture (95:5, v/v) for protein precipitation and BHT (butylated hydroxy toluene) as stabilizer for sample preparation. Both analytes were analyzed on Kromasil 100 C₁₈ column (150 mm × 4.6 mm, 5 μm), Brownlee analytical (Perkin Elmer) C₁₈ column (150 mm × 4.6 mm, 5 μm), and Supelco (Supelcosil) LC-18 column (150 mm × 3 mm, 3 μm), protected by a Perkin Elmer C₁₈ (30 mm × 4.6 mm, 10 μm; Norwalk, USA) pre-column guard cartridge, at 292 nm wavelength, using methanol–water (99:1, v/v), in isocratic mode as mobile phase applied at flow rate of 1.5 ml/min and 1 ml/min for both 5 μm and 3 μm columns, respectively. Complete separation of all the analytes was achieved in 3 and 6 min on 3 μm and 5 μm columns, respectively by injecting 20 μl of sample into the HPLC system by autosampler, keeping column oven temperature at 25 °C. Different particulate reversed-phase chromatographic columns were evaluated in order to select the best column in terms of sensitivity, selectivity, resolution and short run time of both the analytes and it was concluded that 3 μm columns are better to be used in clinical set up as well as in laboratories for the separation of these analytes in a shorter time as compared with 5 μm columns. The method was validated and applied for the analysis of all-trans-retinol and α-tocopherol in the serum of human volunteers.     

Dimethyl sulfoxide-induced hydroxyapatite formation: a biological model of matrix vesicle nucleation to screen inhibitors of mineralization

To elucidate the inhibition mechanisms of hydroxyapatite (HA), a biological model mimicking the mineralization process was developed. The addition of 4% (v/v) dimethyl sulfoxide (DMSO) in synthetic cartilage lymph (SCL) medium containing 2 mM calcium and 3.42 mM inorganic phosphate (P_i) at pH 7.6 and 37 °C produced HA as matrix vesicles (MVs) under physiological conditions. Such a model has the advantage of monitoring the HA nucleation process without interfering with other processes at the cellular or enzymatic level. Turbidity measurements allowed us to follow the process of nucleation, whereas infrared spectra and X-ray diffraction permitted us to identify HA. Mineral formation induced by DMSO and by MVs in the SCL medium produced crystalline HA in a similar manner. The nucleation model served to evaluate the inhibition effects of ATP, GTP, UTP, ADP, ADP-ribose, AMP, and pyrophosphate (PP_i). Here 10 μM PP_i, 100 μM nucleotide triphosphates (ATP, GTP, UTP), and 1 mM ADP inhibited HA formation directly, whereas 1 mM ADP-ribose and 1 mM AMP did not. This confirmed that the PP_i group is a potent inhibitor of HA formation. Increasing the PP_i concentration from 100 μM to 1 mM induced calcium pyrophosphate dihydrate. We propose that DMSO-induced HA formation could serve to screen putative inhibitors of mineral formation.     

Determination of TJ0711 hydrochloride in rat plasma by high performance liquid chromatography with fluorescence detection and its application to pharmacokinetics

In the present study, a simple and sensitive high performance liquid chromatography with fluorescence detection (HPLC-FD) method was developed to determine TJ0711 hydrochloride, a novel α- and β-receptor blocker. TJ0711 hydrochloride and verapamil hydrochloride (the internal standard) were separated on Knauer Eurospher C₁₈ (250 mm × 4.0 mm i.d., 5 μm) column at 50 °C. The mobile phase was methanol:perchloric acid (12 nM, aq) (56:44, v:v), with a flow rate of 1.0 mL/min. The wavelengths of FD were set at 246 nm for excitation and 300 nm for emission. For plasma samples of rats, the analytes were extracted with acetic ether from alkalinized plasma, and then back-extracted into 10 mM dilute sulfuric acid. The linearity was over a concentration range of 20–10,000 ng/mL. The intra- and inter-day precisions referred by relative standard deviation were less than 2.0% and 4.3%, respectively. The mean analytical recoveries of TJ0711 hydrochloride at different concentrations (50, 1000 and 8000 ng/mL) ranged from 88.3% to 92.9%. The lower limit of quantification (LLOQ) was 20 ng/mL. Finally, this method was successfully applied to the estimation of pharmacokinetic parameters of TJ0711 hydrochloride after intravenous doses of 4, 8 and 16 mg/kg in rats.