Optimization of the separation conditions of tetracyclines on a preselected reversed-phase column with embedded urea group

The use of a C12 stationary phase with embedded polar group has been investigated for the separation of seven tetracyclines. The influence of pH, organic modifier, buffer, and temperature on the peak shape and analyte separation was discussed. It appears that all the chromatographic conditions had a great effect on both the resolution and peak shape whereas the elution order was not affected. The baseline separation with symmetrical peaks of the seven tetracyclines can be obtained with a mobile phase containing either 5 mM phosphate buffer pH 2.5/ACN (84:16 v/v) or 5 mM perchlorate buffer pH 2.5/ACN (75:25 v/v) at a temperature not exceeding 20 degrees C. This study reveals that the retention mechanism is ion-pairing.     

Capillary electrochromatography of biologically relevant flavonoids

Flavonoids were separated utilizing CEC technique. Baseline separation of biologically relevant flavonoids was obtained using a 100 microm ID fused-silica capillary filled with 3 microm Silica-C18 material and an optimized mobile phase comprising of 20 mM Tris-HCl (pH 6.5), ACN and water at a ratio of 10/40/50 v/v/v. Separations were carried out at 25 kV and a column temperature of 25 degrees C. The influence of relevant parameters for the CEC separation, such as buffer concentration, pH, separation voltage, and ACN concentration, was investigated and optimized. Dependencies of the electroendoosmotic flow (EOF) on these parameters and effects on the resolution of the analytes were studied. During analyses the solvents used for dissolving the samples turned out to have significant effects on the separation of flavonoids. The optimized system was then successfully used for the separation of the flavonoids epicatechin, myricetin, quercetin, naringenin, and hesperetin. CEC turned out to be a useful complementary tool for the economic analysis of flavonoids in addition to common HPLC, muHPLC, and CE methodologies. This method can be used for real applications in phytomics.     

Effect of stability of internal standard on quantification of 15 flavonoids in Epimedium using CZE

A CZE method was developed for the simultaneous determination of 15 flavonoids, including epimedin B, epimedin A, hexandraside F, epimedin C, icariin, sagittatoside B, sagittatoside A, hexandraside E, 2′′‐O‐rhamnosyl icariside II, baohuoside VII, baohuoside I, caohuoside C, epimedoside C, baohuoside II, and kaempferol‐3‐O‐rhamnoside, in different species of Epimedium, and the effect of stability of internal standard (IS) on quantification was also investigated. As a result, rutin was not available for use as an IS because of its unstable property in sample solution, which suggested that the stability of IS both in standards and sample solution should be considered for the analysis. Using stable daidzein as IS, the analysis was performed within 35 min by using 50 mM borax buffer containing 20% ACN as a modifier (pH 10.0), while separation voltage was 25 kV and temperature was at 30°C. The method was validated to be accurate, simple, and repeatable, and was successfully applied to the analysis of 36 samples from 17 species of Epimedium.     

Micellar electrokinetic and high-performance liquid chromatographic determination of potential manufacturing impurities in pholcodine

Quantitative high-performance liquid chromatographic (HPLC) and micellar electrokinetic chromatographic (MEKC) methods have been developed for the determination of four structurally related potential manufacturing impurities, including morphine, of the opiate derivative pholcodine. Pholcodine and the four impurities were separated by MEKC in less than 14 min using a 70 cm x 75 microm I.D. uncoated fused-silica capillary (25 kV at 30 degrees C) and a running buffer consisting of 10% acetonitrile (v/v) in 20 mM borate-phosphate buffer pH 8.0 containing 40 mM sodium dodecyl sulphate (SDS). The MEKC method was compared to a HPLC method using a 5 microm Luna phenyl-hexyl column (150 x 4.6 mm I.D.) eluted with a mobile phase consisting of a mixture of 10% (v/v) acetonitrile, 7% (v/v) tetrahydrofuran in 20 mM phosphate buffer pH 8.0. Both methods were fully validated and a comparison was made regarding selectivity, linearity, precision, robustness and limits of detection and quantitation. The presence of the impurities in different samples of pholcodine drug substance was investigated using both methods.     

Chiral capillary electrophoretic determination of the enantiomeric purity of homocamptothecin derivatives, promising antitumor topoisomerase I inhibitors, using highly sulfated CDs and fluorescence detection

EKC methods for the enantiomeric resolution of homocamptothecin derivatives, potent anticancer agents targeting DNA topoisomerase I selected for clinical trials, were developed using highly sulfated beta-CD as chiral selectors at acidic pH. Optimal electrophoretic conditions, with migration times under 15 min, were as follows: for the neutral homocamptothecin analog 1, a BGE of 75 mM phosphate buffer pH 2.5 (H(3)PO(4) + triethanolamine)/ACN - 95/5 v/v, with 7.5% w/v highly S-beta-CD, an applied field of 0.2 kV/cm and a fused capillary temperature control of 30 +/- 0.1 degrees C (typical current approximately 175 microA); for the cationic homocamptothecin 2, a BGE of 25 mM phosphate buffer pH 2.5 (H(3)PO(4) + TEA)/ACN - 90/10 v/v, with 2.5% w/v highly S-beta-CD, an applied field of 0.15 kV/cm and a fused capillary temperature control of 25 +/- 0.1 degrees C (typical current approximately 45 muA), and both are validated. The best results in terms of LOQ were obtained by EC with fluorescence detection: 10 ng/mL and 20 ng/mL for 1 and 2, respectively (LOQ divided by 150 for 1 and 5 for 2 with respect to UV), thus making this method particularly convenient for enantiomeric purity determination of galenic forms. UV detection appears to be an alternative to fluorescence for the analysis of the main component either for the control of galenic forms or for therapeutic adaptation. Moreover, this method exhibits better performances than HPLC.     

Determination of the enantiomeric impurity in S-(-)pantoprazole using high performance liquid chromatography with sulfobutylether-beta-cyclodextrin as chiral additive

A new and accurate HPLC method using sulfobutylether-beta-cyclodextrin (SBE-beta-CD) as chiral mobile phase additive (CMPA) was developed and validated for the determination of R-(+)pantoprazole in S-(-)pantoprazole. The influences of type and concentration of CD, ACN content and buffer pH of mobile phase on the resolution and retention of enantiomers were investigated. A baseline resolution of pantoprazole enantiomers was achieved on a Spherigel C18 column (150 mm x 4.6 mm, 5 microm) using ACN and 10 mM phosphate buffer (pH 2.5) containing 10 mM SBE-beta-CD (15:85 v/v) as mobile phase with a flow rate of 0.9 mL/min at 20 degrees C. The detection wavelength was set at 290 nm. The method was extensively validated in terms of accuracy, precision and linearity according to the International Conference on Harmonisation (ICH) guidelines and proved to be robust. The LOD and LOQ for R-(+)pantoprazole were 0.2 and 0.5 microg/mL, respectively, with 5 microL injection volume. A good linear relationship was obtained in the concentration range of 0.5-6.0 microg/mL with r(2) >0.999 for R-(+)pantoprazole. The percentage recovery of the R-(+)pantoprazole ranged from 92.1 to 101.2 in bulk drug of S-(-)pantoprazole. The method is capable of determining a minimum limit of 0.05% w/w of R-enantiomer in S-(-)pantoprazole bulk samples.     

Simultaneous determination of 15 flavonoids in Epimedium using pressurized liquid extraction and high-performance liquid chromatography

Herba Epimedii (family Berberidaceae), Yinyanghuo in Chinese, is one of the commonly used Chinese medicines. Flavonoids are considered as its active components. In this study, a reliable pressurized liquid extraction (PLE) and HPLC method was developed for simultaneous determination of 15 flavonoids, namely hexandraside E, kaempferol-3-O-rhamnoside, hexandraside F, epimedin A, epimedin B, epimedin C, icariin, epimedoside C, baohuoside II, caohuoside C, baohuoside VII, sagittatoside A, sagittatoside B, 2'-O-rhamnosyl icariside II and baohuoside I in different species of Epimedium. The analysis was performed by using a Zorbax SB-C18 analytical column (250 mm x 4.6 mm I.D., 5 microm) at gradient elution of water and acetonitrile with diode-array detection (270 nm). All calibration curves showed good linearity (r(2)>0.9997) within test ranges. The LOD and LOQ were lower than 1.31 ng and 2.62 ng on column, respectively. The RSD for intra- and inter-day of 15 analytes was less than 3.8% at three levels, and the recoveries were 90.5-106.8%. The validated method was successfully applied for the analysis of 15 flavonoids in different species of Epimedium which had great variation on the contents of investigated flavonoids. Hierarchical clustering analysis based on the characteristics of 15 investigated compound peaks in HPLC profiles showed that 26 samples were divided into three main clusters, which were in accordance with their flavonoid contents. Four flavonoids including epimedin A, B, C and icariin were optimized as markers for quality control of the species of Epimedium used as Yinyanghuo.     

Development and validation of capillary electrophoresis method for tobramycin with precapillary derivatization and UV detection

One of the major drawbacks in the analysis of aminoglycoside antibiotics is their lack of UV chromophore and/or fluorophore. Tobramycin, a representative member of this group, was examined in this study. To overcome the detection hurdle, a precapillary derivatization followed by capillary electrophoresis analysis with direct UV detection was investigated. A central composite design was applied to optimize the method and three parameters were selected in this study: buffer pH, temperature and % acetonitrile (ACN). Selectivity between tobramycin main component and its adjacent peaks as well as the peak efficiency and symmetry factors were established as responses. For each response, a model was obtained by a second-order mathematical expression. Successful results were obtained with a simple background electrolyte (BGE) containing 30 mM sodium tetraborate, pH 10.2, and ACN (75:25 v/v). Under these conditions, baseline separation of tobramycin from its adjacent kanamycin B and an unknown peak was achieved. A temperature of 20 degrees C and applied voltage of 28.0 kV were used. The method showed good validation data in terms of precision, limits of quantitation and detection, specificity and linearity and was found to be suitable for analysis of tobramycin bulk pharmaceutical samples.     

CEC separation of insect oostatic peptides using a strong-cation-exchange stationary phase

The separation of several insect oostatic peptides (IOPs) was achieved by using CEC with a strong-cation-exchange (SCX) stationary phase in the fused-silica capillary column of 75 microm id. The effect of organic modifier, ionic strength, buffer pH, applied voltage, and temperature on peptides' resolution was evaluated. Baseline separation of the studied IOPs was achieved using a mobile phase containing 100 mM pH 2.3 sodium phosphate buffer/water/ACN (10:20:70 v/v/v). In order to reduce the analysis time, experiments were performed in the short side mode where the stationary phase was packed for 7 cm only. The selection of the experimental parameters strongly influenced the retention time, resolution, and retention factor. An acidic pH was selected in order to positively charge the analyzed peptides, the pI's of which are about 3 in water buffer solutions. A good selectivity and resolution was achieved at pH <2.8; at higher pH the three parameters decreased due to reduced or even zero charge of peptides. The increase in the ionic strength of the buffer present in the mobile phase caused a decrease in retention factor for all the studied compounds due to the decreased interaction between analytes and stationary phase. Raising the ACN concentration in the mobile phase in the range 40-80% v/v caused an increase in both retention factor, retention time, and resolution due to the hydrophilic interactions of IOPs with free silanols and sulfonic groups of the stationary phase.     

Preparation and evaluation of C18-bonded 1-microm silica particles for pressurized capillary electrochromatography

Nonporous silica spheres (1 microm) were synthesized and bonded with octadecylsilane functionality. These stationary phase particles were packed electrokinetically into fused-silica capillaries with 100 microm id for a length of 20 cm, which was evaluated by using pressurized CEC (pCEC). The efficiency of the C18 RP column was characterized through the theoretical plates of thiourea, benzyl alcohol, toluene, styrene, and naphthalene. The effects of experimental parameters such as the applied voltage, sample size, pump flow rate, pH value and the concentration of the buffer solution, and the content of methanol in the mobile phase, on-column efficiency were evaluated. Column efficiency as high as 200 000 theoretical plates per meter for naphthalene was obtained with the optimal condition of 70% v/v methanol and 30% v/v of 10 mmol/L phosphate buffer (pH 7.8) at an applied voltage of 10 kV and a supplementary pressure of 500 psi.     

Determination of residual fluoroquinolones in honey by liquid chromatography using metal chelate affinity chromatography

A new analytical method for the simultaneous determination of seven fluoroquinolones, namely, norfloxacin, ciprofloxacin, danofloxacin, enrofloxacin, orbifloxacin, sarafloxacin, and difloxacin, especially in dark-colored honey, has been developed. Fluoroquinolone antibiotics were extracted from samples with MacIlvaine buffer solution (pH 4.0) containing EDTA disodium salt dihydrate. The extracts were treated with both a polymeric cartridge and a metal chelate affinity column preloaded with ferric ion (Fe3+). LC separation with fluorescence detection was performed at 40 degrees C using an Inertsil ODS-4 analytical column (150 x 4.6 mm, 3 microm). The mobile phase was composed of 20 mM/L citrate buffer solution (pH 3.1)-acetonitrile mixture (70 + 30, v/v) containing 1 mM/L sodium dodecyl sulfate. Lomefloxacin was used as an internal standard. The developed method was validated according to the criteria of European Commission Decision 2002/657/EC. Decision limits and detection capabilities were below 2.9 and 4.4 microg/kg, respectively.     

Micellar electrokinetic chromatography method for the determination of sulfamethoxazole, trimethoprim and their main metabolites in human serum

A complete analytical procedure, including sample clean-up and a micellar electrokinetic chromatographic method, is presented for the determination of sulfamethoxazole, trimethoprim, and their main metabolites by using 20 mmol L(-1) borate buffer (pH 9.3), 25 mmol L(-1) sodium dodecylsulfate, and 5% v/v acetonitrile as electrolyte. The separation was carried out at 30 kV and 20 degrees C in a fused silica capillary (60.2 cm x 75 microm inner diameter) fitted with a window in the capillary cartridge of 100 x 800 microm. The detector response was linear from the limit of quantification to 3 mg L(-1) for the individual components. The limits of quantification ranged from 0.13 up to 0.24 mg L(-1). The method was applied to human serum, previously spiked at different concentrations of all the analytes, and recoveries between 95% and 108% were obtained.     

Development of a capillary zone electrophoresis method for the separation of a furan combinatorial library

Nine component mixtures of a furan library were simultaneously separated by capillary zone electrophoresis (CZE) using a phosphate buffer as a background electrolyte at low pH. The effects of buffer concentration, buffer pH, type and concentration of organic solvents on the electrophoretic mobility, resolution, and analysis time were systematically investigated. Resolution and efficiency of furan library components were further improved using cyclodextrin (CD)-modified CZE. Under optimum conditions, eight of the nine furans were baseline-resolved in less than 10 min at 30 kV using 50 mM phosphate buffer, 10% v/v acetonitrile (ACN), pH 2.0, with 5 mM gamma-CD.     

Micellar electrokinetic chromatography of polychlorinated biphenyl congeners using a polymeric surfactant as the pseudostationary phase

Micellar electrokinetic chromatography (MEKC) of highly hydrophobic compounds is generally difficult using sodium dodecyl sulfate micellar solutions. The polymeric surfactant, polysodium undecyl sulfate (poly-SUS) has been used to separate moderately to highly hydrophobic polychlorinated biphenyl (PCB) congeners by MEKC in the absence of cyclodextrins. Parameters such as concentration of acetonitrile (ACN), polymeric surfactant concentration, and the effect of pH were examined. Optimum MEKC conditions to get baseline resolution of nine PCBs was 7.5 mM borate in 40% (v/v) ACN fraction buffered at pH 9.2 using 0.5% (w/v) poly-SUS. The applied voltage was 30 kV and the temperature was maintained at 25 degrees C. Elution order for each PCB congener was found to be dependent on the degree of chlorination and hydrophobic character.     

Development and validation of an HPLC method for determination of ziprasidone and its impurities in pharmaceutical dosage forms

Ziprasidone is known as a novel "atypical" or "second-generation" antipsychotic drug. A sensitive and reproducible method was developed and validated for determination of ziprasidone and its major impurities, which are significantly different in polarity. The separation is performed on a Waters Spherisorb octadecylsilyl 1 column (5.0 microm particle size, 250 x 4.6 mm id) using a gradient with mobile phase A [buffer-acetonitrile (80+20, v/v)] and mobile phase B [buffer-acetonitrile (10+90, v/v)] at a working temperature of 25 degrees C. The buffer was 0.05 M KH2PO4 solution with an addition of 10 mL triethylamine/L solution, adjusted to pH 2.5 with orthophosphoric acid. The flow rate was 1.5 mL/min, and the eluate was monitored at 250 nm using a diode array detector. Optimization of the experimental conditions was performed using partial least squares regression, for which four factors were selected for optimization: buffer concentration, buffer pH, triethylamine concentration, and temperature. The proposed validated method is convenient and reliable for the assay and purity control in both raw materials and dosage forms.     

Reversed-phase capillary electrochromatography for the simultaneous determination of acetylsalicylic acid, paracetamol, and caffeine in analgesic tablets

The separation and simultaneous determination of caffeine, paracetamol, and acetylsalicylic acid in two analgesic tablet formulations was investigated by capillary electrochromatography (CEC). The effect of mobile phase composition on the separation and peak efficiency of the three analytes was studied and evaluated; in particular, the influence of buffer type, buffer pH, and acetonitrile content of the mobile phase was investigated. The analyses were carried out under optimized separation conditions, using a full-packed silica capillary (75 microm ID; 30.0 cm and 21.5 cm total and effective lengths, respectively) with a 5 microm C8 stationary phase. A mixture of 25 mM ammonium formate at pH 3.0 and acetonitrile (30:70 v/v) was used as the mobile phase. UV detection was at 210 nm. Good linearity was found in the range of 50-200, 20-160, and 4-20 microg/mL for acetylsalicylic acid (r2=0.9988), paracetamol (r2=0.9990) and caffeine (r2=0.9990), respectively. Intermediate precision (RSD interday) as low as 0.1-0.8% was found for retention times, while the RSD values for the peak area ratios (Aanalyte/AIS) were in the range of 1.9-2.9%. The optimized CEC method was applied to the analysis of the studied compounds present in commercial tablets.     

Optimization via experimental design of an SPE-HPLC-UV-fluorescence method for the determination of valsartan and its metabolite in human plasma samples

A chemometric approach was applied for the optimization of the extraction and separation of the antihypertensive drug valsartan and its metabolite valeryl-4-hydroxy-valsartan from human plasma samples. Due to the high number of experimental and response variables to be studied, fractional factorial design (FFD) and central composite design (CCD) were used to optimize the HPLC-UV-fluorescence method. First, the significant variables were chosen with the help of FFD; then, a CCD was run to obtain the optimal values for the significant variables. The measured responses were the corrected areas of the two analytes and the resolution between the chromatographic peaks. Separation of valsartan, its metabolite valeryl-4-hydroxy-valsartan and candesartan M1, used as internal standard, was made using an Atlantis dC18 100 mm x 3.9 mm id, 100 angstroms, 3 microm chromatographic column. The mobile phase was run in gradient elution mode and consisted of ACN with 0.025% TFA and a 5 mM phosphate buffer with 0.025% TFA at pH 2.5. The initial percentage of ACN was 32% with a stepness of 4.5%/min to reach the 50%. A flow rate of 1.30 mL/min was applied throughout the chromatographic run, and the column temperature was kept to 40+/-0.2 degrees C. In the SPE procedure, experimental design was also used in order at achieve a maximum recovery percentage and extracts free from plasma interferences. The extraction procedure for spiked human plasma samples was carried out using C8 cartridges, phosphate buffer (pH 2, 60 mM) as conditioning agent, a washing step with methanol-phosphate buffer (40:60 v/v), a drying step of 8 min, and diethyl ether as eluent. The SPE-HPLC-UV-fluorescence method developed allowed the separation and quantitation of valsartan and its metabolite from human plasma samples with an adequate resolution and a total analysis time of 1 h.     

Micellar electrokinetic chromatography of fluorescently labeled proteins on poly(dimethylsiloxane)-based microchips

MEKC of standard proteins was investigated on PDMS microfluidic devices. Standard proteins were labeled with AlexaFluor(R) 488 carboxylic acid tetrafluorophenyl ester and filtered through a size-exclusion column to remove any small peptides and unreacted label. High-efficiency MEKC separations of these standard proteins were performed using a buffer consisting of 10 mM sodium tetraborate, 25 mM SDS, and 20% v/v ACN. A separation of BSA using this buffer in a 3.0 cm long channel generated a peak with a plate height of 0.38 microm in <20 s. Additional fast separations of myoglobin, alpha-lactalbumin, lysozyme, and cytochrome c also yielded peaks with plate heights ranging from 0.54 to 0.72 microm. All proteins migrated with respect to their individual pIs. To improve the separations, we used a PDMS serpentine chip with tapered turns and a separation distance of 25 cm. The number of plates generated increased linearly with increasing separation distance on the extended separation channel chips; however, the resolution reached an asymptotic value after about 7 cm. This limited the peak capacity of the separation technique to 10-12.     

Optimization of a stability-indicating HPLC method for the simultaneous determination of rifampicin, isoniazid, and pyrazinamide in a fixed-dose combination using artificial neural networks

The aim of this study is to develop and optimize a simple and reliable high-performance liquid chromatography (HPLC) method for the simultaneous determination of rifampicin (RIF), isoniazid (INH), and pyrazinamide (PZA) in a fixed-dose combination. The method is developed and optimized using an artificial neural network (ANN) for data modeling. Retention times under different experimental conditions (solvent, buffer type, and pH) and using four different column types (referred to as the input and testing data) are used to train, validate, and test the ANN model. The developed model is then used to maximize HPLC performance by optimizing separation. The sensitivity of the separation (retention time) to the changes in column type, concentration, and type of solvent and buffer in the mobile phase are investigated. Acetonitrile (ACN) as a solvent and tetrabutylammonium hydroxide (tBAH), used to adjust pH, have the greatest influence on the chromatographic separation of PZA and INH and are used for the final optimization. The best separation and reasonably short retention times are produced on the micro-bondapak C18, 4.6 x 250-mm column, 10 microm/125 A using ACN-tBAH (42.5:57.5, v/v) (0.0002M) as the mobile phase, and optimized at a final pH of 3.10.     

Analysis of tricyclic antidepressant drugs in plasma by means of solid-phase microextraction-liquid chromatography-mass spectrometry

Solid-phase microextraction coupled to liquid chromatography and mass spectrometry (SPME-LC-MS) was used to analyze tricyclic antidepressant drugs desipramine, imipramine, nortriptyline, amitriptyline, and clomipramine (internal standard) in plasma samples. SPME was performed by direct extraction on a PDMS/DVB (60 microm) coated fiber, employing a stirring rate of 1200 rpm for 30 min, pH 11.0, and temperature of 30 degrees C. Drug desorption was carried out by exposing the fiber to the liquid chromatography mobile phase for 20 min, using a labmade SPME-LC interface at 50 degrees C. The main variables experimentally influencing LC-MS response were evaluated and mathematically modeled. A rational optimization with fewer experiments was achieved using a factorial design approach. The constructed empirical models were adjusted with 96-98% of explained deviation allowing an adequate data set comprehension. The chromatographic separation was realized using an RP-18 column (150 mm x 2.1 mm, 5 microm particles) and ammonium acetate buffer (0.01 mol/l, pH 5.50) : acetonitrile (50 : 50 v/v) as mobile phase. Low detection levels were achieved with electrospray interface (0.1 ng/ml). The developed method showed specificity, linearity, precision, and limit of quantification adequate to assay tricyclic antidepressant drugs in plasma.