Simultaneous micellar liquid chromatographic analysis of seven water-soluble vitamins: optimization using super-modified simplex

A super-modified simplex (SMS) method has been used to optimize the mobile phase used for separation of seven water-soluble vitamins in multivitamin tablets by gradient micellar liquid chromatography (MLC) with ultraviolet (UV) detection at 254, 295, and 361 nm. Effect of column temperature and addition of organic modifier to the mobile phase on separation efficiency were investigated: the appropriate conditions used were a temperature of 35 degrees C and 1-butanol modifier. The sodium dodecyl sulfate (SDS) concentration, pH, and 1-butanol% in the mobile phase were chosen for simultaneous optimization using the SMS method. The optimum mobile phase was found to be 16 mmol L(-1) (mM) SDS, 0.02 M phosphate buffer, pH 3.6, and a gradient of 3.5-10% (v/v) butanol. The total analysis time for vitamins was 75 min. The analytical parameters including linearity ( r>0.9970), limit of detection (0.12-50 micro g mL(-1)), precision of method (relative standard deviation (RSD) <8.90%), and accuracy obtained by the recovery assay (88-103%) support the usefulness of the proposed method for the determination of the water-soluble vitamins.     

Fast HPLC method using ion-pair and hydrophilic interaction liquid chromatography for determination of phenylephrine in pharmaceutical formulations

A rapid procedure based on a direct extraction and HPLC determination with fluorescence detection of phenylephrine in pharmaceutical sachets that include a large excess of paracetamol (65 + 1, w/w), ascorbic acid (5 + 1, w/w), and other excipients (aspartame and sucrose) was developed and validated. The final optimized chromatographic method for ion-pair chromatography used an XTerra RP18 column, 3 microm particle size, 50 x 3.0 mm id. The mobile phase consisted of a mixture of acetonitrile and buffer (10 mM sodium octane-1-sulfonate, adjusted with H3PO4 to pH 2.2; 200 + 800, v/v), with a constant flow rate of 0.3 mL/min. The separation was carried out at 30 degrees C, and the injection volume was 3 microL. Fluorescence detection was performed at excitation and emission wavelengths of 275 and 310 nm, respectively. The mobile phase parameters, such as the organic solvent fraction (acetonitrile) in mobile phase as an organic modifier, the concentration of sodium octane-1-sulfonate as a counter-ion, temperature, and pH of mobile phase, were studied. As an alternative to ion-pair chromatography, hydrophilic interaction liquid chromatography (HILIC) was investigated using a Luna HILIC column, 3 microm, 100 x 4.6 mm id. The mobile phase consisted of acetonitrile and buffer (5 mM potassium dihydrogen phosphate, adjusted with H3PO4 to pH 2.5; 750 + 250, v/v) at a flow rate of 0.8 mL/min. The separation was carried out at 25 degrees C, and the injection volume was 5 microL. The proposed method has an advantage of a very simple sample pretreatment, and is much faster than the currently utilized HPLC methods using gradient elution and UV detection. Commercial samples of sachets were successfully analyzed by the proposed HPLC method.     

Preconcentration based on paramagnetic microparticles for the separation of sarcosine using hydrophilic interaction liquid chromatography coupled with coulometric detection

Sarcosine has been identified as a potential prostate cancer marker. To provide determination of this compound, a number of methods are developing. In this study, we optimized a method for its separation by hydrophilic interaction LC with electrochemical detection (ED). Due to the fact that mobile phases commonly used for this type of separation altered the LODs measured by electrochemical detectors, we applied postcolumn dosing of buffer suitable for ED. The optimized conditions were mobile phase A acetonitrile, mobile phase B water in the ratio A/B 70:30, with postcolumn addition of mobile phase C (200 mM phosphate buffer pH 9). The optimal mixing ratio was A + B/C 1:1 with a flow rate of 0.80 mL/min (0.40 + 0.40 mL/min) and detection potential of 1000 mV. Due to the optimization of the parameters for effective separation, which had to meet the optimal parameters of ED, we reached a good resolution for separation also with a good LOD (100 nM). In addition, we successfully carried out sarcosine analysis bound on our modified paramagnetic microparticles with the ability to preconcentrate sarcosine isolated from artificial urine.     

Simultaneous estimation of acetylsalicylic acid and clopidogrel bisulfate in pure powder and tablet formulations by high-performance column liquid chromatography and high-performance thin-layer chromatography

This paper describes validated high-performance column liquid chromatographic (HPLC) and high-performance thin-layer chromatographic (HPTLC) methods for simultaneous estimation of acetylsalicylic acid (ASA) and clopidogrel bisulfate (CLP) in pure powder and formulations. The HPLC separation was achieved on a Nucleosil C8 column (150 mm length x 4.6 mm id, 5 microm particle size) using acetonitrile-phosphate buffer, pH 3.0 (55 + 45, v/v) mobile phase at a flow rate of 1.0 mL/min at ambient temperature. The HPTLC separation was achieved on an aluminum-backed layer of silica gel 60F254 using ethyl acetate-methanol-toluene-glacial acetic acid (5.0 + 1.0 + 4.0 + 0.1, v/v/v/v) mobile phase. Quantitation was achieved with UV detection at 235 nm over the concentration range 4-24 microg/mL for both drugs, with mean recoveries of 99.98 +/- 0.28 and 100.16 +/- 0.66% for ASA and CLP, respectively, using the HPLC method. Quantitation was achieved with UV detection at 235 nm over the concentration range of 400-1400 ng/spot for both drugs, with mean recoveries of 99.93 +/- 0.55 and 100.21 +/- 0.83% for ASA and CLP, respectively, using the HPTLC method. These methods are simple, precise, and sensitive, and they are applicable for the simultaneous determination of ASA and CLP in pure powder and formulations.     

Fast separation and sensitive detection of carcinogenic aromatic amines by reversed-phase micro-liquid chromatography coupled with electrochemical detection

A micro-LC method was developed for the fast and sensitive analysis of aromatic amines by electrochemical detection. The chromatographic separation of nine carcinogenic aromatic amines was performed on an ABZ + PLUS column with detection limits up to pM L(-1) levels. Mobile phase comprised of methanol-acetate buffer of pH 5 (45:55, v/v) used at a flow rate of 0.2 ml min(-1). The detection was performed with a 6 mm glassy carbon electrode at an applied potential of 0.8 V versus Ag/AgCl. An intraday RSD for retention time and peak area were between 0.22% and 0.73% and 1.86% and 4.03%, respectively. The interdays RSD for retention time and peak area were between 0.47% and 1.35% and 2.04% and 4.42%, respectively. The applicability of the assay has been demonstrated by analyzing these aromatic amines in lake water and synthetic food colour additives. A comparison is given between electrochemical and UV detection.     

Determination of Benzocaine Using HPLC and FIA with Amperometric Detection on a Carbon Paste Electrode

A new method for benzocaine determination employing FIA and HPLC with electrochemical detection on a carbon paste electrode was developed. The optimum conditions for the determination were found. Carrier solution for FIA consisted of B–R buffer pH 4 (80 % methanol, v/v) and used flow rate was 1.0 mL min^?1. Mobile phase for HPLC consisted of B–R buffer pH 4 (75 % methanol, v/v) with flow rate 0.4 mL min^?1. Working potential of +1.2 V was employed. Practical applicability of the methods was tested on the determination of benzocaine in selected pharmaceuticals. The results were in agreement with results obtained using spectrophotometric detection and with one exception also with the content declared by the manufacturer.     

Determination of tryptophan and kynurenine in human plasma by liquid chromatography-electrochemical detection with multi-wall carbon nanotube-modified glassy carbon electrode

A novel method was developed for the simultaneous determination of kynurenine and tryptophan by high‐performance liquid chromatography with electrochemical detection at multi‐wall carbon nanotube (MWCNT)‐modified glassy carbon electrode. The separation and detection conditions were optimized. The typical HPLC experiments were conducted by using a reversed‐phase ODS column with a mobile phase consisting of stock acetate buffer (pH 5)–methanol (4:1, v/v) using an isocratic elution at the flow rate of 1.0 mL/min. The obtained LODs for kynurenine and tryptophane were 0.5 and 0.4 µmol/L, respectively. The analytical method for human plasma samples was validated and confirmed by LC‐UV and LC‐MS. The recoveries were in the range of 84.8–110%, and the precision was lower than 5.9%. Copyright © 2010 John Wiley & Sons, Ltd.     

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.     

Quantitation of glucosamine from shrimp waste using HPLC

This work presents a high-performance liquid chromatography (HPLC) method for the quantitation of glucosamine in chitin. The method includes an acid hydrolysis of chitin. The chromatographic separation is achieved using a Hypersil ODS 5-microm column (250 x 4.6 mm) at 38 degrees C, with precolumn derivatization with 9-fluorenylmethyl-chloroformate and UV detection (lambda = 264 nm). The mobile phase is a mixture of mobile phase A [30 mM ammonium phosphate (pH 6.5) in 15:85 methanol-water (v/v)], mobile phase B [15:85 methanol-water (v/v)], and mobile phase C [90:10 acetonitrile-water (v/v)], with a flow rate of 1.2 mL/min. The HPLC method proposed showed adequate repeatability (relative standard deviation, 5.8%), accuracy (92.7% recovery), and sensitivity, with a detection limit of 2 microg/mL. The method is successfully applied to the quantitation of glucosamine for the determination of the purity of chitin from shrimp waste.     

On-line coupling of pressurized capillary electrochromatography with end-column amperometric detection for analysis of estrogens

An improved technique, pressurized capillary electrochromatography (pCEC) coupling with end-column amperometric detection (AD), was developed and used for the separation and determination of estrogens. The effects of pH value, composition of mobile phase, concentration of the surfactant sodium dodecyl sulfate (SDS) and applied voltage on separation were investigated. The electrochemical oxidation of diethylstilbestrol (DES), dienestrol (DE), and hexestrol (HEX) could be reliably monitored with a carbon electrode at 0.9 V (vs. Ag/AgCl). The pCEC analyses were performed on a capillary separation column packed with 3 microm C18 particles with an acetonitrile/water (31%: 69%) mobile phase containing Tris buffer (5 mmol/L, pH 4.5) and 4 mmol/L SDS. High voltage up to 12 kV reduced the retention time dramatically and still provided a baseline resolution. In addition, supplementary pressure prevented bubble formation and provided reliability and reproducibility of the pCEC performance. The detection limits for the three estrogens ranged from 1.2 to 2.2x10(-7) mol/L, about 10 20-fold lower than those obtained with pCEC-UV detection. To evaluate the feasibility and reliability of this system, the proposed pCEC-AD method was further demonstrated with fish muscle samples spiked with estrogens.     

Reverse-phase liquid chromatographic determination of α-lipoic acid in dietary supplements using a boron-doped diamond electrode

A fast liquid chromatographic separation, coupled with sensitive and straightforward detection using a boron-doped diamond (BDD) electrode, was developed and validated for the determination of α-lipoic acid in dietary supplement samples. The analysis was carried out using a reversed phase C18 (150 mm × 4.6 mm, 5 μm) column with a mobile phase consisting of a 1:1 (v/v) ratio of 0.05 M phosphate solution (pH 2.5):acetonitrile, at a flow rate of 1.0 mL/min. The detection potential obtained from hydrodynamic voltammetry was 1.05 V vs. Ag/AgCl. Under optimized conditions, the chromatographic separation was performed in less than 5 min, a good linear relationship was obtained between the current and the α-lipoic concentration within the range of 0.01-60 μg/mL (correlation coefficient of 0.9971), and a detection limit of 3.0 ng/mL was determined. Furthermore, this method was successfully applied to determine α-lipoic acid concentrations in selected commercial dietary supplement samples. The recovery of α-lipoic acid in spiked samples at 0.5, 5.0 and 30 μg/mL ranged from 94.4% to 103.6% with a relative standard deviation (RSD) of between 1.2% and 3.7%. In real samples, this developed methodology produced results that were highly correlated with the standard HPLC-UV approach. Therefore, the present method can be used for fast, selective and sensitive quantification of α-lipoic acid in dietary supplements.     

Determination of rutin and forsythin in fruit ofForsythia suspensa(Thunb.) Vahl by capillary electrophoresis-electrochemical detection

Summary Capillary electrophoresis-amperometric detection is evaluated for simultaneous determination of rutin and forsythin. The cyclic voltammogram, hydrodynamic voltammogram, effect of pH, buffer concentration and SDS, and percent organic modifier on separation and detection were studied. Conditions were optimized as follows: 1.2 V detection potential; separation at 12 kV; 5 s at 15 kV for sample injection time and sample injection voltage; mobile phase 20 mM boric acid buffer; pH 8.4, containing 40 mM SDS and 10% (v/v) acetontrile. The method gave low detection limit as 0.001 mg mL⁻¹ and 0.0005 mg mL⁻¹ (S/N=3), wide linear range 0.005–0.5 mg mL⁻¹ for rutin and forsythin, respectively. The relative standard deviations of peak current and migration time for 8 consecutive injections of the standard solution containing 0.1 mg mL⁻¹ each compound were 4.78%, 3.63% and 6.40%, 2.95% for rutin and forsythin, respectively. In addition, levels of the two compounds in traditional Chinese herbal drugs were easily determined.     

Microemulsion electrokinetic chromatography versus capillary electrochromatography-UV-mass spectrometry for the analysis of flunitrazepam and its major metabolites

Benzodiazepines, namely flunitrazepam and its three major metabolites, were successfully separated by microemulsion electrokinetic chromatography. Separation was achieved using an untreated fused-silica capillary (48 cm (effective length 40 cm) x 50 num) at 25 kV; detection was performed by UV at 220 nm. The microemulsion system consisted of 70 mM octane, 800 mM 1-butanol, 80 mM sodium dodecyl sulfate (SDS) and 10 mM borate buffer, pH 9. Very high efficiencies (up to 400 000 plates) and resolution better than 3 were achieved. Since this technique is not compatible with mass spectrometry (MS) detection, a capillary electrochromatographic (CEC) method was developed to separate flunitrazepam and its metabolites. The effects of mobile phase composition and pH as well as voltage and temperature were systematically investigated. The optimized CEC method allowed the baseline separation of the investigated compounds. For the on-line coupling of CEC with electrospray ionization-mass spectrometry, the column was connected to a void fused-silica capillary using a Teflon connection. This configuration was found efficient and suitable for hyphenation of commercial CEC and MS instrumentation using commercially available CEC columns.     

Improved efficiency in micellar liquid chromatography using triethylamine and 1-butanol as mobile phase additives to reduce surfactant adsorption

The effect of triethylamine as a mobile phase modifier on chromatographic efficiency in micellar liquid chromatography (MLC) is reported for nine different columns with various bonded stationary phases and silica pore sizes, including large-pore short alkyl chain, non-porous, and perfluorinated. Reduced plate height (h) versus reduced velocity (nu) plots were constructed for each column and the A' and C' terms calculated using a simplified Van Deemter equation introduced in our previous work. To further explore the practicality of using triethylamine in the micellar mobile phase, the efficiency of nine polar and non-polar substituted benzenes was studied on seven columns. Surfactant adsorption isotherms were measured for five columns with three micellar mobile phases to understand the relationship between adsorbed surfactant, mobile phase additive, and column efficiency. Clear improvements in efficiency were observed with the addition of 2% (v/v) triethylamine to a 1-butanol modified aqueous micellar mobile phase. This finding is supported by the lower amount of surfactant adsorbed onto the stationary phase when TEA is present in the mobile phase compared to an SDS only or a 1-butanol modified SDS mobile phase.     

Determination of total cholesterol in serum by high-performance liquid chromatography with electrochemical detection

A simple and sensitive HPLC method that does not require derivatization for determining cholesterol has been developed. Investigation of voltammetric behavior of cholesterol showed that cholesterol could be oxidized on a glassy carbon electrode in non-aqueous solvents. This was applied to the development of a method by HPLC with electrochemical detection (HPLC-ED). The HPLC-ED was optimized using the separation of cholesterol and oxysterols including 26-hydroxycholesterol and 24S-hydroxycholesterol. The separation was carried out with a Develosil C30-UG-3 column; acetonitrile-2-propanol (9:1, v/v) containing 50mM LiClO(4) as a mobile phase; and an applied potential at 1.9V versus Ag/AgCl. The current peak height was linearly related to the amount of cholesterol injected from 0.5-100 microM (r>0.999). The detection limit (S/N=3) of cholesterol was 0.36 microM (1.8 pmol). Cholesterol at 100 microM was directly detected with a relative standard deviation (RSD) of less than 1.0% (n=8). Total cholesterol and free cholesterol in control human serum were determined by the present method with the recovery of more than 90% and the RSD (n=6) of less than 3.0%.     

Determination of picogram amounts of lipoxin A4 and lipoxin B4 by high-performance liquid chromatography with electrochemical detection

A new method for the determination of lipoxins with electrochemical detection after high-performance liquid chromatography is described. The half-wave potentials of lipoxin A4 and lipoxin B4 at a glassy carbon electrode and a mobile phase of methanol-water (65:35 v/v) and 1 mM trifluoroacetic acid was found to be +1.14 V versus an Ag/AgCl reference electrode. The use of trifluoroacetic acid instead of sulphuric acid and lithium perchlorate led to a background current of 6-8 nA at +1.20 V. The detection limits for both lipoxins, based on a signal-to-noise ratio of 3:1 were found to be 5-10 pg (15-30 fmol). The new method was applied to an extract of human polymorphonuclear granulocytes, preincubated with 15-hydroxyeicosatetraenoic acid and stimulated with Ca2+ ionophore A23187.     

Separation of structurally related estrogens using isocratic elution pressurized capillary electrochromatography

In this paper, the pressurized capillary electrochromatography (pCEC) with UV detection was utilized for the separation and determination of three structurally related estrogens, such as diethylstilbestrol (DES), hexestrol (HEX) and dienestrol (DE), which were difficult to be separated by capillary electrophoresis (CE) and HPLC due to their similarity in the structure and charge-to-mass ratios. Experiments were carried out in a commercially available pCEC instrument using a capillary column packed with 3 microm octadecyl silica (ODS). Surfactant sodium dodecyl sulfate (SDS) was introduced in the mobile phase to enhance the speed of analysis. The effective factors on the retention time and separation resolution, such as the applied voltage, supplementary pressure, the pH and the concentration of the buffer solution, the concentration of SDS, and the content of acetonitrile in the mobile phase, were evaluated. Based on the investigation, 31% (v/v) acetonitrile and 69% (v/v) of 10 mmol/L phosphate buffer (pH 6.5) containing 1.0 mmol/L SDS at an applied voltage of -12 kV and a supplementary pressure of 1000 psi were found to be the optimal conditions for pCEC to separate the three estrogens. The method also had been applied to the analysis of fish muscle samples spiked with estrogens.     

Separation and detection of common mono- and divalent cations by ion chromatography with an ODS column and conductivity/UV detection

The retention and detection behavior of common mono- and divalent cations (M⁺, alkali metal (Li⁺, Na⁺, K⁺, Rb⁺, Cs⁺) and ammonium ions (NH₄⁺); M²⁺, alkaline earth metal ions (Mg²⁺, Ca²⁺, Sr²⁺, Ba²⁺) was examined using an ODS column (150×4.6 mm I.D.) and conductivity (CD)/UV detection. The results obtained were as follows: (1) for M⁺, the mobile phase, 0.1 mM sodium dodecyl sulphate (SDS)+10 mM HNO₃ and indirect CD detection were effective. (2) Addition of Ce(III) in the mobile phase accelerated the elution of both M⁺ and M²⁺. The separation of above 10 cations on an ODS column was achieved for the first time without any coelution of cations and disturbance by system peak. Addition of higher SDS resulted in good separation of M⁺ and M²⁺ with longer retention times. CD detection was possible for M⁺ and M²⁺ and UV detection for M²⁺. (3) For M²⁺, the mobile phase, 0.8 mM Ce(III)+0.1 mM SDS+1 mM HNO₃ and indirect UV detection were effective. The IC methods were applied to real samples.     

Analysis of flunarizine in the presence of some of its degradation products using micellar liquid chromatography (MLC) or microemulsion liquid chromatography (MELC)--application to dosage forms

The separation of flunarizine hydrochloride (FLZ) and five of its degradation products--1-[bis(4-fluorophenyl)methyl]-4-(3-phenyl-2-propenyl)piperazine, 4-oxide (A), bis(4-fluorophenyl)methanone (B), bis(4-fluorophenyl)methanol (C), 1-(3-phenyl-2-propenyl)piperazine(D), and 1-[bis-4-fluorophenyl) methyl] piperazine (E)--could be accomplished by reversed phase liquid chromatography using either micellar or microemulsion mobile phases. Cyanopropyl-bonded stationary phase has been used with UV detection at 254 nm. Microemulsion mobile phase consisting of 0.15 M SDS, 10% n-propanol, 1% n-octanol, and 0.3% triethylamine in 0.02 M phosphoric acid of pH 7.0, has been used for the separation of FLZ and its degradation products (B, C, D, and E). Micellar mobile phases consisting of 0.15 M sodium dodecyl sulphate (SDS), 10% n-propanol, 0.3% triethylamine (TEA) in 0.02 M phosphoric acid of pH values either 4.0 or 6.8 have been used for the separation of FLZ from its degradation products, i.e. either from (B, C, D, and E) or from (A, B, C, and D), respectively. Micellar liquid chromatography (MLC) was applied to the determination of FLZ in pure form as well as in dosage forms; the calibration graph was linear over the concentration range of 0.15-50 microg/mL with detection limit of 0.02 microg/mL (4.19 x 10(-8)M).     

Comparison of HPLC and MEEKC for Miconazole Nitrate Determination in Pharmaceutical Formulation

A simple solid phase extraction (SPE) method coupled with high performance liquid chromatography (HPLC) using UV detector and microemulsion electrokinetic chromatography (MEEKC) has been developed and compared for the quantitative determination of miconazole nitrate in pharmaceutical formulation. For HPLC method, two parameters were optimized, namely, the wavelength and the mobile phases. The optimized condition was at the 225 nm wavelength and the mobile phase of ACN:MeOH (90:10 v/v). There are seven MEEKC parameters that were optimized, in this research, which were applied to voltage, temperature, wavelength, sodium dodecyl sulfate (SDS) concentration, buffer pH, buffer concentration and butan-1-ol concentration. The optimum MEEKC condition was obtained using 86.35 % (w/w) 2.5 mM borate buffer pH 9, 0.25 % (w/w) SDS, 0.8 % (w/w) ethyl acetate, 6.6 % w/w butan-1-ol and 6.0 % (w/w) acetonitrile. The combination of SPE using a diol column with HPLC–UV and the MEEKC methods were successfully applied for the determination of miconazole nitrate in a pharmaceutical formulation with the recovery percentage of 98.35 and 92.50 %, respectively.