高选择性高效液相色谱-电化学检测法测定丹参中5种酚类物质

发展了一种高灵敏度的高效液相色谱-电化学检测（HPLC-ECD）方法,用于丹参中5种酚类物质的定量分析。为了获得较高的检测灵敏度,对流动相的pH、缓冲溶液类型和浓度、有机相种类和梯度以及流速做了系统研究。在较低的pH（2.8）、较低的缓冲盐浓度（20 mmol/L NaH₂PO₄）和较缓的乙腈梯度下,以0.2 mL/min流速可为5种酚类的分离检测提供较好的分离度和较高的检测灵敏度。在获得优化的分离参数后,将其用于14批丹参药材中5种酚类物质的定量分析。结果表明,该方法可获得较好的回收率（>95%）、较宽的线性范围（高达4个数量级）、较好的重复性（RSD<4.01%）和较高的灵敏度（咖啡酸的LOQ低至1.5μg/L）。与紫外检测方法相比,ECD检测方法具有更高的选择性,可减少非抗氧化活性的物质的干扰。     

Rational experimental design for bioanalytical methods validation illustration using an assay method for total captopril in plasma

Generally, bioanalytical chromatographic methods are validated according to a predefined programme and distinguish a pre-validation phase, a main validation phase and a follow-up validation phase. In this paper, a rational, total performance evaluation programme for chromatographic methods is presented. The design was developed in particular for the pre-validation and main validation phases. The entire experimental design can be performed within six analytical runs. The first run (pre-validation phase) is used to assess the validity of the expected concentration-response relationship (lack of fit, goodness of fit), to assess the specificity of the method and to assess the stability of processed samples in the autosampler for 30 h (benchtop stability). The latter experiment is performed to justify overnight analyses. Following approval of the method after the pre-validation phase, the next five runs (main validation phase) are performed to evaluate method precision and accuracy, recovery, freezing and thawing stability and over-curve control /dilution. The design is nested, i.e., many experimental results are used for the evaluation of several performance characteristics. Analysis of variance (ANOVA) is used for the evaluation of lack of fit and goodness of fit, precision and accuracy, freezing and thawing stability and over-curve control/ dilution. Regression analysis is used to evaluate benchtop stability. For over-curve control/ dilution, additional to ANOVA, also a paired comparison is applied. As a consequence, the recommended design combines the performance of as few independent validation experiments as possible with modern statistical methods, resulting in optimum use of information. A demonstration of the entire validation programme is given for an HPLC method for the determination of total captopril in human plasma.     

应用质量源于设计的理念优化大鼠血浆中大黄蒽醌的分析方法

应用质量源于设计理念建立一种高效液相色谱-荧光检测法(HPLC-FLD)用于测定大鼠血浆中5种大黄蒽醌。用Plackett-Burman设计考察流动相中甲醇含量、pH值、流速、柱温和进样体积对色谱峰的分离度、理论塔板数、最末洗脱峰的保留时间和拖尾因子的影响,结果显示流动相中甲醇含量、流速和柱温对色谱系统的影响显著(p<0.05)。继而采用Box-Behnken设计结合响应面法考察上述三因素对分离度、保留时间和理论塔板数的影响。用Derringer渴求函数评价了响应值的综合作用。得出最优色谱条件为:以甲醇-0.1%(v/v)磷酸水溶液(81.4:18.6, v/v)为流动相等度洗脱,流速1.1 mL/min,柱温31℃,荧光检测激发波长为440 nm,发射波长为540 nm。建立的模型显示良好的预测性。结果表明:质量源于设计的理念可有效地应用于优化高效液相色谱分析方法。     

Validation of a high-performance liquid chromatographic method for determination of isoflavonoids in soybeans. Study of the extraction procedure by experimental design

Summary An improved analytical method, reversed-phase high-performance liquid chromatography on a narrow-bore C₁₈ column, has been developed for the simultaneous determination of genistein, daidzein, formononetin, and biochanin A. The method was validated in terms of detection limits, quantitation limits (LOQ), linearity, and precision.LOQ in the 0.04–0.1 μg mL⁻¹ range were calculated, enabling determination of these compounds of nutritional concern at trace levels. Good linearity was demonstrated over three orders of magnitude of concentration for each analyte (r ²=0.998–1.000). The intra-day repeatability was evaluated in terms ofRSD (%) at two concentration levels for each analyte (RSD (%) <1.8%). Good inter-day reproducibility of data was proved by performing homoscedasticity and ANOVA tests (P>0.05 at the 95% confidence level). The method was applied to the determination of genistein and daidzein in yellow soybeans, after optimization of the method for extraction of isoflavonoid aglycones from soybeans by experimental design, i.e. central composite design. Extraction recoveries up to 87±4% were obtained when the corresponding glycosidic forms (genistin and daidzin) were added to soybean samples.     

Optimization of temperature-controlled ionic liquid dispersive liquid phase microextraction combined with high performance liquid chromatography for analysis of chlorobenzenes in water samples

Temperature-controlled ionic liquid dispersive liquid phase microextraction (TCIL-DLPME) combined with high performance liquid chromatography-diode array detection (HPLC-DAD) was applied for preconcentration and determination of chlorobenzenes in well water samples. The proposed method used 1-butyl-3-methylimidazolium hexafluorophosphate ([C₄mim][PF₆]) as the extraction solvent. The effect of different variables on extraction efficiency was studied simultaneously using an experimental design. The variables of interest in the TCIL-DLPME were extraction solvent volume, salt effect, solution temperature, extraction time, centrifugation time, and heating time. The Plackett-Burman design was employed for screening to determine the variables significantly affecting the extraction efficiency. Then, the significant factors were optimized by using a central composite design (CCD) and the response surface equations were developed. The optimal experimental conditions obtained from this statistical evaluation included: extraction solvent volume, 75 μL; extraction time, 20 min; centrifugation time, 25 min; heating time, 4 min; solution temperature, 50 °C; and no addition of salt. Under optimal conditions, the preconcentration factors were between 187 and 298. The limit of detections (LODs) ranged from 0.05 μg L⁻¹ (for 1,2-dichlorobenzene) to 0.1 μg L⁻¹ (for 1,2,3-trichlorobenzene). Linear dynamic ranges (LDRs) of 0.5-300 and 0.5-500 μg L⁻¹ were obtained for dichloro- and trichlorobenzenes, respectively. The performance of the method was evaluated for extraction and determination of chlorobenzenes in well water samples in micrograms per liter and satisfactory results were obtained (RSDs < 9.2%).     

高灵敏、非衍生的高效液相色谱-电化学检测方法定量分析环维黄杨星D

发展了一种检测血液和环维黄杨星D（CVB-D）药片中CVB-D的高效液相色谱-电化学检测方法（HPLC-ECD）。由于使用高灵敏度的掺硼金刚石电极（BDD）、可为碱性化合物提供更好峰形的C18HCE色谱柱和优化的流动相,该方法可提供更高的检测灵敏度,检出限（LOD）和定量限（LOQ）分别为0.198和0.297μg/L。该方法灵敏度比紫外（UV）、蒸发光散射（ELSD）、电雾式检测（CAD）和质谱（MS）方法灵敏度分别高12727、11481、2630和16.8倍。同时,该方法可提供较宽的线性范围（0.297~1891μg/L）,并且操作过程比MS方法更简单。该方法用于低质量浓度（59.1μg/L）样品的检测也可以提供较好的日内（峰面积RSD<5.08%）和日间（峰面积RSD<5.57%）重复性。此外,将该方法稍做修改,还可用于其他碱性化合物的检测。     

Electrokinetic extraction on artificial liquid membranes of amphetamine-type stimulants from urine samples followed by high performance liquid chromatography analysis

Electromembrane extraction (EME) coupled with high performance liquid chromatography and ultraviolet detection was developed for determination of amphetamine-type stimulants in human urine samples. Amphetamines migrated from 3 mL of different human urine matrices, through a thin layer of 2-nitrophenyl octyl ether (NPOE) containing 15% tris-(2-ethylhexyl) phosphate (TEHP) immobilized in the pores of a porous hollow fiber, and into a 15 μL acidic aqueous acceptor solution present inside the lumen of the fiber. Equilibrium extraction conditions were obtained after 7 min of operation. Experimental design and response surface methodology (RSM) were used for optimization of EME parameters. Under optimal conditions, amphetamines were effectively extracted with recoveries in the range of 54-70%, which corresponded to preconcentration factors in the range of 108-140. The calibration curves were investigated in the range of 0-7 μg mL(-1) and good linearity was achieved with a coefficient of estimation better than 0.991. Detection limits and inter-day precision (n=3) were less than 0.01 μg mL(-1) and 11.2%, respectively.     

Science based calibration for the extraction of 'analyte-specific' HPLC-DAD chromatograms in environmental analysis

Multivariate science based calibration (SBC) has been applied to the resolution of overlapped peaks in liquid chromatography with diode array detection (LC-DAD). Complex river water samples spiked with 11 pharmaceutical substances resulted in poorly resolved chromatograms containing additional peaks from interfering matrix compounds and a change in the background absorbance due to the mobile phase gradient. Applying the present multivariate approach it was possible to resolve all 11 analytes from overlapping peaks, obtaining linear calibration lines (R² > 0.96). Recovery percentages on spiked samples ranged between 74.6 and 113.5%, which are quite satisfactory taking into account the low concentration ranges considered to 1-7 μg L⁻¹.     

Phase optimized liquid chromatography as an instrument for steroid analysis

A new method using phase optimized LC (POPLC) for the analysis of steroids is described. The retention factors and the theoretical plate numbers of different steroids were determined for four different stationary phases. Based on these values, an optimal stationary phase composition and the resultant chromatogram have been calculated by an optimization software and compared to the measured data. It is demonstrated that these predicted data show only little differences to the experimental results. Furthermore, it is shown that the overall selectivity of the optimal stationary phase composition is much better than the selectivity of any individual stationary phase.     

微透析活体取样与高效液相色谱-电化学检测法联用测定帕金森大鼠脑中神经递质

建立了一种微透析活体取样与高效液相色谱-电化学检测法联用技术测定自由活动大鼠脑中7种单胺类神经递质的方法.高效液相色谱采用Aglient XDB-C18柱,流动相为pH 3.0的0.1 mol/L H3PO4-NaH2PO4缓冲液与甲醇的混合液(90:10,V/V),流速为0.3 mL/min,电化学检测的工作电极为玻...     

Optimization of alcohol‐assisted dispersive liquid–liquid microextraction by experimental design for the rapid determination of fluoxetine in biological samples

A sensitive and rapid method based on alcohol‐assisted dispersive liquid–liquid microextraction followed by high‐performance liquid chromatography for the determination of fluoxetine in human plasma and urine samples was developed. The effects of six parameters on the extraction recovery were investigated and optimized utilizing Plackett–Burman design and Box–Benken design, respectively. According to the Plackett–Burman design results, the volume of disperser solvent, extraction time, and stirring speed had no effect on the recovery of fluoxetine. The optimized conditions included a mixture of 172 μL of 1‐octanol as extraction solvent and 400 μL of methanol as disperser solvent, pH of 11.3 and 0% w/v of salt in the sample solution. Replicating the experiment in optimized condition for five times, gave the average extraction recoveries equal to 90.15%. The detection limit of fluoxetine in human plasma was obtained 3 ng/mL, and the linearity was in the range of 10–1200 ng/mL. The corresponding values for human urine were 4.2 ng/mL with the linearity range from 10 to 2000 ng/mL. Relative standard deviations for intra and inter day extraction of fluoxetine were less than 7% in five measurements. The developed method was successfully applied for the determination of fluoxetine in human plasma and urine samples.     

Application of an optimized dispersive nanomaterial ultrasound‐assisted microextraction method for preconcentration of carbofuran and propoxur and their determination by high‐performance liquid chromatography with UV detection

An extraction method based on dispersive nanomaterial ultrasound‐assisted microextraction was used for the preconcentration of carbofuran and propoxur insecticides in water samples prior to high‐performance liquid chromatography with UV detection. ZnS:Ni nanoparticles were synthesized based on the reaction of the mixture of zinc acetate and nickel acetate with thioacetamide in aqueous media and then loaded on activated carbon (ZnS:Ni‐AC). Different methods were used for recognizing the properties of ZnS:Ni‐AC and then this nanomaterial was used for extraction of carbamate insecticide as new adsorbent. The influence of variables on the extraction method (such as amount of adsorbent (mg: NiZnS‐AC), pH and ionic strength of sample solution, vortex and ultrasonic time (min), ultrasound temperature and desorption volume (mL) was investigated by a screening 2^7–4 Plackett–Burman design. Then the significant variables were optimized by using a central composite design combined with a desirability function. At optimum conditions, this method had linear response >0.0060–10 μg/mL with detection limit 0.0015 μg/mL and relative standard deviations <5.0% (n = 3).     

Applying green analytical chemistry for development and validation of RP-HPLC stability indicating assay method for estimation of fenoverine in bulk and dosage form using quality by design approach

A green and robust reverse-phase liquid chromatographic method has been developed for the determination of fenoverine (FEN), by applying combined principles of green analytical chemistry and quality by design approaches on a Spherisorb C18 column (150?×?4.6?mm, 3?µm) with UV detection at 262?nm. A two level fractional factorial design (2^^7-3) Res IV was used for screening of influential chromatographic factors. The critical method parameters actively affecting critical quality attributes (CQAs) were identified and further optimized using Box–Behnken design. The predicted optimum assay conditions comprised of methanol and ammonium acetate buffer 20?mM, in an extent of 81:19% v/v individually having a flow rate of 1.0?mL/min with a column oven temperature of 33°C. The drug was stressed in hydrolytic, oxidative, reductive, thermal, and photolytic conditions. The developed method was validated successfully. The detector response was linear in the concentration of 0.5–160?µg/mL with a limit of detection (LOD) and limit of quantitation (LOQ) as 0.1 and 0.3?µg/mL, respectively. The % recovery was found to be 99.7%. The analytical method volume intensity value for developed method was 45?mL and the environment assessment tool (EAT) score was 41.07. The method is simple, environmentally benign, rapid, and robust for the determination of FEN in bulk and in its dosage form.     

Determination of Mycotoxins Using Hollow Fiber Dispersive Liquid–Liquid–Microextraction (HF-DLLME) Prior to High-Performance Liquid Chromatography – Tandem Mass Spectrometry (HPLC - MS/MS)

A modified hollow-fiber-supported dispersive liquid-liquid microextraction (HF-DLLME) method was developed for the determination of aflatoxins and ochratoxin A in food samples. The various parameters affecting the efficiency of extraction, such as pH, salt addition, extraction time, stirring rate, desorption time, type and volume of extractant and disperser solvents were carefully studied and optimized using two step strategies. The linearity of the evaluated results was 0.1 to 30?μg L^?1 for aflatoxins and 0.1 to 20?μg L^?1 for ochratoxin A, with regression coefficients (R²) exceeding 0.9990. The precision was satisfactory with relative standard deviation values less than 11%. The method accuracy was within the recommended range from 70% to 120% and analyte accuracy between 83% and 101%. The limits of detection and quantification were in the range from 0.04 to 0.06?μg L^?1 and 0.08 to 0.13?μg L^?1, respectively, for multi-aflatoxins, and 0.02 to 0.04?µg L^?1 and 0.08 to 0.10?µg L^?1, respectively, for ochratoxin A. The developed method was successfully applied for the determination of mycotoxins in food samples.     

Recovery of H2SO4 from wastewater in the presence of NaCl and KHCO3 through pH responsive polysulfone membrane: Optimization approach

Through the phase inversion technique, asymmetric flat sheet pH-responsive Polysulfone (PSF) membrane was prepared and utilized for recovering H₂SO₄ in the presence of NaCl and KHCO₃ from wastewater. Hydrophilic and pH-responsive characteristics were incorporated within the membrane by blending Polyethylene glycol methyl ether (PEGME) and Humic acid (HA). The modification in membrane morphology with pH was characterized by Field Emission Scanning Electron Microscopy (FESEM), Differential scanning calorimetry (DSC) and Fourier Transform Infrared Studies (FTIR) method. The ion exchange capacity of the prepared pH-responsive membrane increased from 0.145 to 0.25 mmol/g when compared to the pristine PSF membrane. Pure water flux (PWF) of 113.8–46.8 L/m²h, water uptake of 25.9%–6.8% were obtained for pH-responsive membrane when pH varied from 4 to 12. Recovery of H₂SO₄ was optimized by design expert software 9.0 TRIAL and was found to be a maximum of 76.57 ± 1.5% in the presence of 0.32 M NaCl and 0.5 M KHCO₃ at pH ~8.4, through the pH-responsive PSF membrane by diffusion dialysis process. The influencing parameters (pH, NaCl (M) and KHCO₃ (M)) were optimized and acid recovery modeling was performed through response surface methodology (RSM) and central composite design (CCD). F value of 6573.40 through ANOVA study indicated the significance of the quadratic model chosen, whereas an insignificant lack of fit (prob > F = 0.0519) confirmed the goodness of fit between the model and obtained experimental data's.     

HPLC-PDA analysis of ACE-inhibitors,hydrochlorothiazide and indapamide utilizing design of experiments

A simple, rapid, precise, accurate and sensitive high performance liquid chromatographic method has been developed for simultaneous determination of ACE inhibitors with hydrochlorothiazide and indapamide in pharmaceutical formulations. ‘Design of Experiments’ (DoE) using ‘central composite design’ (CCD) was applied to facilitate method development and optimization. Mobile phase was optimized utilizing response surface methodology using Design Expert software. Chromatographic separation was achieved on Hypersil®-Gold C₁₈ (100 × 4.6 mm, 3 μm, Thermo Fisher Scientific, USA), column at 25 °C. The mobile phase was 58% buffer (5 mM KH₂PO₄, containing triethylamine 0.25 ml/L), 25% acetonitrile and 17% methanol (pH adjusted to 2.8 ± 0.1). The analysis was performed at 215 nm. The mobile phase flow rate was 1.0 ml/min and injection volume 10 μl. The method was validated for linearity, limits of quantitation and detection, accuracy, precision, ruggedness and robustness as per the International Conference on Harmonization (ICH) guidelines. Calibration curves (for lisinopril, hydrochlorothiazide, captopril, imidapril, perindopril, indapamide and trandolapril) were linear in the concentration range of 5–35 μg/ml. The limit of detection and limit of quantitation for experimental drugs ranged from 0.03 to 0.61 and 0.08–1.84 μg/ml respectively.     

Statistical experimental design for bioprocess modeling and optimization analysis

The statistical design of experiments (DOE) is a collection of predetermined settings of the process variables of interest, which provides an efficient procedure for planning experiments. Experiments on biological processes typically produce long sequences of successive observations on each experimental unit (plant, animal, bioreactor, fermenter, or flask) in response to several treatments (combination of factors). Cell culture and other biotech-related experiments used to be performed by repeated-measures method of experimental design coupled with different levels of several process factors to investigate dynamic biological process. Data collected from this design can be analyzed by several kinds of general linear model (GLM) statistical methods such as multivariate analysis of variance (MANOVA), univariate ANOVA (timesplit-plot analysis with randomization restriction), and analysis of orthogonal polynomial contrasts of repeated factor (linear coefficient analysis). Last, regression model was introduced to describe responses over time to the different treatments along with model residual analysis. Statistical analysis of biprocess with repeated measurements can help investigate environmental factors and effects affecting physiological and bioprocesses in analyzing and optimizing biotechnology production.     

A novel sorptive extraction method based on polydimethylsiloxane frit for determination of lung cancer biomarkers in human serum

In this study, a porous polypropylene frit was coated with polydimethylsiloxane (PDMS) as extraction medium, based on the home-made PDMS-frit, a rapid, simple and sensitive sorptive extraction method was established for analysis of potential biomarkers of lung cancer (hexanal and heptanal) in human serum samples. In the method, derivatization and extraction occurred simultaneously on the PDMS-frit, then the loaded frit was ultrasonically desorbed in acetonitrile. Polymerization, derivatization–extraction and desorption conditions were optimized. Under the optimal conditions, satisfactory results were gained, a wide linear application range was obtained in the range of 0.002–5.0 μmol L⁻¹ (R > 0.997) for two aldehydes, the detection limits (S N⁻¹ = 3) were 0.5 nmol L⁻¹ for hexanal and 0.4 nmol L⁻¹ for heptanal. The relative standard deviations (RSDs, n = 5) of the method were below 7.9% and the recoveries were above 72.7% for the spiked serum. All these results hint that the proposed method is potential for disease markers analysis in complex biological samples.     

Hydrophilic interaction chromatography for the analysis of aminoglycosides

The effect of mobile-phase constituents (pH and ionic strength) and chromatographic behaviour of ten aminoglycosides (streptomycin, dihydrostreptomycin, spectinomycin, apramycin, paramomycin, kanamycin A, gentamycin C1, gentamycin C2/C2a, gentamycin C1a and neomycin) in the bare silica, amino, amide and zwitterionic phases of hydrophilic interaction chromatography (HILIC) were studied systematically. Among the stationary phases studied, the zwitterionic phase provided the best separation of aminoglycosides. The effect of pH, ionic concentration and column temperature on retention time, peak shape and sensitivity was studied using a central composite design. pH affected sensitivity of the detection of analytes but not the retention time. High ionic strength in the mobile phase was necessary to control the ionic interactions between ionised aminoglycosides and the hydrophilic phase, thereby influencing peak shape and retention time. Column temperature affected sensitivity of the detection but not the retention time. During method development, crosstalk between the MS/MS channels of the analytes was observed and resolved.     

Development of an ionic‐liquid‐based dispersive liquid–liquid microextraction method for the determination of antichagasic drugs in human breast milk: Optimization by central composite design

Chagas disease constitutes a major public health problem in Latin America. Human breast milk is a biological sample of great importance for the analysis of therapeutic drugs, as unwanted exposure through breast milk could result in pharmacological effects in the nursing infant. Thus, the goal of breast milk drug analysis is to inquire to which extent a neonate may be exposed to a drug during lactation. In this work, we developed an analytical technique to quantify benznidazole and nifurtimox (the two antichagasic drugs currently available for medical treatment) in human breast milk, with a simple sample pretreatment followed by an ionic‐liquid‐based dispersive liquid–liquid microextraction combined with high‐performance liquid chromatography and UV detection. For this technique, the ionic liquid 1‐octyl‐3‐methylimidazolium hexafluorophosphate has been used as the “extraction solvent.” A central composite design was used to find the optimum values for the significant variables affecting the extraction process: volume of ionic liquid, volume of dispersant solvent, ionic strength, and pH. At the optimum working conditions, the average recoveries were 77.5 and 89.7%, the limits of detection were 0.06 and 0.09 μg/mL and the interday reproducibilities were 6.25 and 5.77% for benznidazole and nifurtimox, respectively. The proposed methodology can be considered sensitive, simple, robust, accurate, and green.