Influence of microwave irradiation on the intraparticle diffusion of an insulin variant in reversed-phase liquid chromatography under linear conditions

The influence of microwave irradiation on the mass transfer kinetics of an insulin variant in reversed-phase liquid chromatography (RPLC) was investigated. The elution band profiles of insulin were obtained by the pulse-response method, under linear conditions. The RPLC column was placed in a microwave oven and the incremental change in the temperature of the column effluent stream at various microwave energies and mobile phase flow rates were measured. The microwave energy dissipated in the column was set at 15 and 30 W and the mobile phase flow rate was varied from 1.0 to 2.5 mL/min at a mobile phase composition of acetonitrile, water, and trifloroacetic acid (31:69:0.1, v/v/v). The experimental data were analyzed using the conventional method of moment analysis and the lumped pore diffusion model. Regardless of mobile flow rates, the effluent temperatures measured at 15 and 30 W microwave power input were 25+/-1 and 30+/-1 degrees C, respectively. The effect of microwave irradiation on the mass transfer of the variant insulin was determined by comparing the band profiles obtained under the same experimental conditions, at the same column temperature, with and without irradiation. The calculated intraparticle diffusion coefficient, D(e), at 30 W (30+/-1 degrees C) microwave irradiation was ca. 20% higher than without irradiation at 30+/-1 degrees C. These preliminary results suggest that microwave irradiation may have a significant influence on the intraparticle diffusion of insulin in RPLC.     

Comparison of chromatographic band profiles obtained under microwave irradiated and non-irradiated reversed-phase liquid chromatography column

The possible influence of the application of microwave energy to a reversed-phase liquid chromatography column on the mass transfer kinetics and the thermodynamics of equilibrium between mobile and stationary phases was examined. Chromatograms of propylbenzene and phenol were recorded under the same experimental conditions, on the same column, successively irradiated and not. The effect of microwave irradiation on the mass transfer kinetics was determined by measuring the second moment of small pulses of propylbenzene in a 70:30 (v/v) solution of methanol in water and microwave outputs of 15 and 30 W. The effect of microwave irradiation on the equilibrium thermodynamics was determined by measuring the elution time of breakthrough curves of phenol at high concentrations in a 20:80 (v/v) solution of methanol and water and microwave outputs of 15, 50, and 150 W. A qualitative comparison of the profiles of the propylbenzene peaks obtained with and without irradiation suggests that this irradiation affects significantly the peak shapes. However, a qualitative comparison of the profiles of the breakthrough curves of phenol obtained with and without irradiation suggests that this irradiation has no significant effect on their shapes. The peak sharpening observed may be due to an increase in the diffusivity, resulting from the dielectric polarization under microwave irradiation. This effect is directly related to an increase of the rate of mass transfers in the column. In contrast, the similarity of the overloaded band profiles at high concentrations suggests that the equilibrium thermodynamics is unaffected by microwave irradiation. This may be explained by the transparence of the stationary phase to microwaves at 2.45 GHz. The column temperature was measured at the column outlet under irradiation powers of 15, 30, 50, and 150 W. It increases with increasing power, the corresponding effluent temperatures being 25+/-1, 30+/-1, 35+/-1, and 45+/-1 degrees C, respectively.     

Influence of the pressure on the properties of chromatographic columns .III. retention volume of thiourea, hold-up volume, and compressibility of the C18-bonded layer

The influence of the average column pressure (ACP) on the elution volume of thiourea was measured on two RPLC columns, packed with Resolve-C18 (surface coverage 2.45 micromol/m2) and Symmetry-C18 (surface coverage 3.18 micromol/m2), and it was compared to that measured under the same conditions on an underivatized silica (Resolve). Five different methanol-water mixtures (20, 40, 60, 80 and 100% methanol, v/v) were used. Once corrected for the compressibility of the mobile phase, the data show that the elution volume of thiourea increases between 3 and 7% on the C18-bonded columns when the ACP increases from 50 to 350 bar, depending on the methanol content of the eluent. No such increase is observed on the underivatized Resolve silica column. This increase is too large to be ascribed to the compressibility of the stationary phase (silica + C18 bonded chains) which accounts for less than 5% of the variation of the retention factor. It is shown that the reason for this effect is of thermodynamic origin, the difference between the partial molar volume of the solute in the stationary and the mobile phase, Delta V, controlling the retention volume of thiourea. While Delta V is nearly constant for all mobile phase compositions on Resolve silica (with Delta V approximately equal to -4 mL/mol), on RPLC phases, it significantly increases with increasing methanol content, particularly above 60% methanol. It varies between -5 mL/mol and -17 mL/mol on Resolve-C18 and between -9 mL/mol and -25 mL/mol on Symmetry-C18. The difference in surface coverage between these two RP-HPLC stationary phases increases the values of Delta V by about 5 mL/mol.     

Simultaneous determination of midazolam and 1'-hydroxymidazolam in human plasma by liquid chromatography with tandem mass spectrometry

A sensitive and simple liquid chromatography-tandem mass spectrometry method for the determination of midazolam and 1'-hydroxymidazolam in human plasma has been developed and validated with a dynamic range of 0.1-250 ng/mL. The analysis was based on semi-automated liquid-liquid extraction followed by evaporation of the extraction solvent, reconstitution and chromatography on a reversed-phase C(18) column. The mobile phase consists of 5 mm ammonium acetate and methanol and runs in gradient at a flow rate of 0.25 mL/min with column temperature of approximately 20 degrees C. The entire column effluent was transferred into the LC-MS/MS interface operated in positive electrospray ionization mode. The chromatographic run time was 4.3 min per injection, with retention times for midazolam, 1'-hydroxymidazolaml and the internal standard, triazolam, of 2.5, 2.3 and 2.1 min, respectively. The intra-day and inter-day precision (RSD %) and accuracy (bias %) of the quality control samples were <15.0% and within +/-13%, respectively. The current method has been applied to a clinical drug-drug interaction study in human.     

环境水样中百菌清残留的单滴微萃取-反相液相色谱测定

应用单滴微萃取(SDME)-反相液相色谱(RPLC)检测了环境水样中的百菌清残留.优化了单滴微萃取条件:环己烷萃取剂6 μL、单滴体积2 μL、搅拌速率350 r/min、萃取时间40 min、水溶液温度35 ℃、无盐度.水样经单滴微萃取后,使用Hypersil C18柱反相液相色谱分离测定百菌清.反相液相色谱条件:100%甲醇流动相、流速1.0 mL/min、柱温25 ℃、224 nm检测.方法的线性范围、检出限、相对标准偏差和富集倍数分别为1.0 ～50 μg/L、0.02 μg/L、6.1%和427倍.采用该法对环境水样中的百菌清残留进行了测定,环境水样的加标回收率为98% ～106%.     

Simultaneous determination of three flavonoid aglycones in Elsholtzia blanda by adopting orthogonal test and high-performance liquid chromatography

A new HCl hydrolysis/HPLC method, by adopting L9(34) orthogonal test to optimize hydrolysis condition, has been developed for simultaneous determination of three flavonoid aglycones in Elsholtzia blanda benth. The HCl concentration, methanol concentration, hydrolysis temperature, hydrolysis time are taking as four inspecting foctors, and the contents of luteolin, apigenin, and 5-hydroxy-6,7-dimethoxyflavone in hydrolytic solution are used as the evaluation indexes. Agilent Zorbax SB-C18 is used as analytical column. The mobile phase is a mixture of methanol-0.2% phosphoric acid (70:30, v/v), and UV detector is set at 350 nm. The flow rate is 1.0 mL/min, the temperature of column is maintained at 30 degrees C. The optimal hydrolysis conditions are 3.0M HCl, 70% methanol, 85 degrees C hydrolytic temperature and 3 h hydrolytic time. Standard curves are linear over the concentration range 8.54-85.4 microg/mL, 1.2-12 microg/mL, 9.2-92 microg/mL, and their average recoveries are 96.8%, 98.0%, and 100.5% for luteolin, apigenin, 5-hydroxy-6, 7-dimethoxyflavone, respectively. Thus, the optimum hydrolysis condition is relatively gentle, and the HPLC method is proved to be simple, accurate, and sensitive, so it will be able be applied to quality control of medicinal plant of Elsholtzia blanda.     

固相萃取技术-HPLC测定复方甘草片中吗啡的含量

报道了用ＳＥＰ－ＰＡＫＣ１８小预柱固相萃取技术和反相高效液相色谱法测定复方甘草片中微量吗啡含量的方法。对固相萃取条件进行了研究,最终选择１０％甲醇溶液为清洗溶液,７０％甲醇溶液为洗脱溶液,基本消除了干扰组分对测定的影响,色谱图背景吸收小,基线平稳。色谱柱为μ－ＢｏｎｄａｐａｋＣ１８柱,流动相为０．１ｍｏｌ／ＬＮａＨ２ＰＯ４溶液－甲醇（５１）,检测波长为２８６ｎｍ。平均回收率为（１０１．２±１．５）％,ＲＳＤ为１．５％。     

Effects of elevated temperature and mobile phase composition on a novel C18 silica column

A novel polydentate C18 silica column was evaluated at an elevated temperature under acidic, basic, and neutral mobile phase conditions using ACN and methanol as the mobile phase organic modifier. The temperature range was 40-200 degrees C. The mobile phase compositions were from 0 to 80% organic-aqueous v/v and the mobile phase pH levels were between 2 and 12. The maximum operating temperature of the column was affected by the amount and type of organic modifier used in the mobile phase. Under neutral conditions, the column showed good column thermal stability at temperatures ranging between 120 and 200 degrees C in methanol-water and ACN-water solvent systems. At pH 2 and 3, the column performed well up to about 160 degrees C at two fixed ACN-buffer compositions. Under basic conditions at elevated temperatures, the column material deteriorated more quickly, but still remained stable up to 100 degrees C at pH 9 and 60 degrees C at pH 10. The results of this study indicate that this novel C18 silica-based column represents a significant advancement in RPLC column technology with enhanced thermal and pH stability when compared to traditional bonded phase silica columns.     

高效液相色谱法测定吲哚美辛控释胶囊的血药浓度和生物利用度

采用ＯＤＳ柱,甲醇－稀磷酸溶液（７６２４）为流动相,２６０ｎｍ为检测波长,建立了测定血浆中吲哚美辛浓度的高效液相色谱法,并测定了吲哚美辛控释胶囊炎痛康的血药浓度。结果表明,血浆中吲哚美辛浓度在０．１２５～５．０ｍｇ／Ｌ范围内线性关系良好（ｒ＝０．９９９６）,检测限６２．５μｇ／Ｌ（Ｓ／Ｎ＝３１）,平均回收率为１００．４％,日内和日间ＲＳＤ均小于５％。１１位受试者单剂量口服炎痛康后的相对生物利用度为１０２．３８％。     

Retention and mass transfer characteristics in reversed-phase liquid chromatography using a tetrahydrofuran-water solution as the mobile phase.

The characteristics of the retention and the mass transfer kinetics in reversed-phase liquid chromatography (RPLC) were measured on a system consisting of a C18-silica gel and a tetrahydrofuran-water (50:50, v/v) solution. These parameters were derived from the first and the second moments of the elution peaks, respectively. Further information on the thermodynamic properties of this system was derived from the temperature dependence of these moments. Some correlations previously established were confirmed for this system, namely, an enthalpy-entropy compensation for both retention and surface diffusion and a linear free-energy relationship. These results are compared with those observed in other similar systems using methanol-water (70:30, v/v) and acetonitnile-water (70:30, v/v) solutions. The contribution of surface diffusion to intraparticle diffusion in C18-silica gel particles was shown to be important. The analysis of the thermodynamic properties of surface diffusion suggests that, in these three RPLC systems, its activation energy is lower than the isosteric heat of adsorption. The nature and the extent of the influence of the mobile phase composition on the parameters describing the retention and the mass transfer kinetics are different but the chromatographic mechanisms involved in RPLC systems appear similar, irrespective of the nature of the organic modifier in the mobile phase.     

Thermostability testing and degradation profiles of doxycycline in bulk, tablets, and capsules by HPLC

A high-performance liquid chromatography (HPLC) method for the quantitation of doxycycline in bulk, tablets, and capsules after storage at -20, 5, 25, 40, 50, 60, and 70 degrees C, has been developed and validated. The samples are eluted from a micro-Bondapak C8 column (4.6x150-mm, i.d., 5-microm particle size) at 27 degrees C, with a mobile phase of acetonitrile-water-THF (29.5:70:0.5, v/v/v), adjusted to pH 2.5 with 1.0M HCl. The flow rate is 1.0 mL/min and detection by UV is at 350 nm. The stability of doxycycline in bulk and in pharmaceuticals is checked over 90 days. Doxycycline shows thermo-degradation after exposure to high temperature; tablets are more stable than capsules. The shelf lives (t90%) are determined to be 1.00, 2.84, and 5.26 years in bulk, capsules, and tablets, respectively, at 25 degrees C. Metacycline and 6-epidoxycyline are identified as degradation products at high temperatures. Amounts of doxycycline, metacycline, and 6-epidoxycycline in all samples are determined by HPLC, and the results compare with those from micellar electrokinetic capillary chromatography. After 90 days, metacycline and 6-epidoxycyline are almost equal in test samples from standard bulk form, tablets, and capsules. It is 27.8+/-0.3%, 13.7+/-0.1%, and 18.8+/-0.2%, respectively.     

An HPLC method for the quantification of sterols in edible seaweeds

This study presents an HPLC method for the quantification of sterols in edible seaweeds. Sterols were identified by HPLC/mass spectrometry (HPLC-MS) in positive APCI mode. The samples were saponified by refluxing with 1 m ethanolic KOH, and the non-saponifiable fraction was extracted with hexane. Sterols were quantified by HPLC with UV detection (HPLC-UV), on a 15 x 0.4 cm Kromasil 100 C(18) 5 micro m column (mobile phase 30:70 v/v methanol:acetonitrile; fl ow rate 1.2 mL/min; column temperature 30 degrees C; detection wavelength 205 nm). Method repeatability for fucosterol was good (coefficient of variation 2.4%). Sterol contents were determined in canned or dried brown seaweeds (Himanthalia elongata, Undaria pinnatifida, Laminaria ochroleuca) and red seaweeds (Palmaria sp., Porphyra sp.). The predominant sterol was fucosterol in brown seaweeds (83-97% of total sterol content; 662-2320 micro g/g dry weight), and desmosterol in red seaweeds (87-93% of total sterol content; 187-337 micro g/g dry weight).     

Chemical stability of midazolam injection by high performance liquid chromatography

The chemical stability of midazolam hydrochloride injection, undiluted or diluted with dextrose sterile solution, was studied at different storage conditions by LC. The study was performed at room temperature (23 +/- 2 degrees C) under light exposure and light protection, +8 +/- 1 degrees C and -20 +/- 0.5 degrees C, in glass and plastic containers over 14 days with midazolam hydrochloride injection, undiluted or diluted with 5% dextrose sterile solution. Chromatographic separation was carried out on a RP-18(e) column, using a mobile phase consisting of ACN-phosphate buffer (pH 3.3; 0.1 M) (30:70 v/v) at a flow rate of 1.0 mL/min and UV detection at 220 nm. The concentrations of all samples remained greater than 90% of the original concentration. The chromatographic assay exhibited an adequate linearity (r(2) >0.999), selectivity, precision (RSD <3.1), and accuracy (recoveries from 100.46 to 101.40%). Injectable midazolam hydrochloride was chemically stable in all conditions that were studied.     

Development and validation of rapid ion-pair RPLC method for simultaneous determination of certain B-complex vitamins along with vitamin C

A rapid, simple, and accurate ion-pair RPLC method has been developed for simultaneous analysis of vitamin C and major B-complex vitamins. An RP C18 column thermostated at 30 degrees C was used with gradient elution of mobile phase comprising 10 mM potassium dihydrogen phosphate buffer (containing 3 mM sodium hexane-1-sulfonate, adjusted to pH 2.80 with o-phosphoric acid) and methanol at a flow rate of 1.0 mL/min to achieve the best possible separation and resolution of all vitamins in about 11.00 min. The detection was performed at 274 nm. The method has been implemented successfully for simultaneous determination of vitamins present in 12 multivitamin/multimineral pharmaceutical preparations, as well as in human urine. Typical validation characteristics were evaluated in accordance with International Conference on Harmonization guidelines. Good linearity over the investigated concentration levels was observed. Intraday repeatability was < or = 2.0%, and interday variation was < or = 2.6%, for all vitamins. The method can be used for assay of these vitamins over a wide concentration range with good precision and accuracy; hence, it would be appropriate for routine QC as well as in clinical analysis.     

用薄层色谱法分离芳香醇胺药物对映体

用D－10-樟脑磷酸铵作为手性离子对试剂添加到流动相中,在硅胶GF254薄层板上分离了两种芳香醇胺类药物对映体。     

Stereoselective determination of hydroxychloro-quine and its major metabolites in human urine by solid-phase microextraction and HPLC

The enantioselective analysis of hydroxychloroquine (HCQ) and its major metabolites was achieved by HPLC and solid-phase microextraction. The chromatographic separation was performed on a Chiralcel OD-H column using hexane/methanol/ethanol (96:2:2, v/v/v) plus 0.2% diethylamine as the mobile phase, at the flow rate of 1.3 mL/min. The main extraction parameters were optimized. The best condition was achieved by the addition of 10% NaCl and 1 mL phosphate buffer 1 mol/L pH 11 to 3 mL human urine. The extraction was conducted for 40 min at 25 degrees C and the desorption time was 3 min using methanol (100%). PDMS-DVB 60 microm fiber was used in this study. The mean recoveries were 9.3, 9.2, and 14.4% for HCQ, desethylhydroxychloroquine (DHCQ), and desethylchloroquine (DCQ), respectively. The method was linear over the range of 50-1000 ng/mL for HCQ enantiomers and over the range of 42-416 ng/mL for DCQ and DHCQ enantiomers. Within-day and between-day precision and accuracy assays for HCQ and its metabolites were lower than 15%. The preliminary 48 h urinary excretion study performed in human urine showed to be stereoselective. The amount of (+)-(S)-enantiomer excreted was higher than its antipode.     

Analysis of semi-volatile organic compounds in aqueous samples by microwave-assisted headspace solid-phase microextraction coupled with gas chromatography-electron capture detection

The pretreatment technique of microwave-assisted headspace solid-phase microextraction (MA-HS-SPME) has been developed and studied for the extraction of semi-volatile organic compounds (SVOCs) in aqueous samples prior to chromatographic analysis. The optimum conditions for obtaining extraction efficiency, such as the extraction time, extraction temperature, addition of salts, and the ratio of sample to headspace volume parameters were investigated. Experimental results indicated that the proposed MA-HS-SPME technique attained the best extraction efficiency under the optimized conditions, i.e., irradiation of extraction solution (20mL aqueous sample in 40mL headspace vial with no addition of salt) under 30W microwave power for 30min at 70 degrees C. The detection was linear at 1-250ng/L with correlation coefficient exceeding 0.997. The detection limits obtained were between 0.2-10.7ng/L, repeatability range from 2 to 15%. Real water samples collected from known sites in southern Taiwan were analyzed using the optimized conditions.     

A simple HPLC method for the quantification of mizoribine in human serum: pharmacokinetic applications

A simple high-performance liquid chromatographic (HPLC) method was developed and validated for the quantification of mizoribine in human serum. After the addition of 70% perchloric acid and 3-methylxanthine (50 microg/mL, internal standard) to human serum, the samples were mixed and centrifuged at 12,000 rpm (1432 g) for 10 min. The supernatant was injected onto a C(18) column eluted with a mobile phase of 20 mm Na2HPO4 and methanol (93:7, v/v, pH 3) containing 0.04% octanesulfonic acid and detected utilizing an ultraviolet detector at 275 nm. The linear calibration curve was obtained in the concentration range of 0.1-4.0 microg/mL and the lower limit of quantification was 0.1 microg/mL. This method was validated with selectivity, linearity, precision and accuracy. In addition, the method was successfully applied to estimate the pharmacokinetic parameters of mizoribine in Korean subjects following an oral administration of 100 mg mizoribine (two Bredinine 50 mg tablets). The maximum serum concentration (C(max)) of 2.30 +/- 0.83 microg/mL was reached 2.27 +/- 0.66 h after an oral dose. The mean AUC(0-12 h) and the elimination half-life (t(1/2)) were 13.2 +/- 4.79 microg h/mL and 3.10 +/- 0.74 h, respectively.     

An improved LC-MS/MS method for quantitative determination of metolazone in human plasma and its application to a pharmacokinetic study

A simple, rapid and accurate liquid chromatography-tandem mass spectrometry method has been developed. After a liquid-liquid extraction procedure, samples were chromatographed on an Agilent TC-C(18) (150 × 4.6 mm, 5 μm) column using an isocratic elution mobile phase composed of methanol and distilled water (70:30, v/v) at a flow rate of 0.5 mL/min. After single-dose administration of 0.5, 1 and 2 mg metolazone, the t(1/2) values were 6.6 ± 2.8, 7.9 ± 1.2 and 7.6 ± 1.9 h, respectively. The pharmacokinetic parameters of multiple doses (1 mg metolazone) were as follows: t(1/2) was 8.9 ± 1.0 h; C(max) was 22.4 ± 5.0 ng/mL; and AUC(0-48) was 156.8 ± 31.6 ng h/mL.