Simultaneous determination of twelve water- and fat-soluble vitamins by high-performance liquid chromatography with diode array detection

Summary A novel method for the simultaneous determination of twelve water- and fat-soluble vitamins has been established by high-performance liquid chromatography with diode array detection. The vitamins were analyzed on a μBondapak C₁₈ column (300 × 3.9 mm, 10 μm) with methanol-KH₂PO₄ buffer (0.1 M, pH 7.0)-water as mobile phase in a gradient. The linearity of calibration graphs was compound-dependent and the detection limits ranged from 0.02 μg mL⁻¹ to 0.5 μg mL⁻¹. The method was successfully applied to determine vitamins in pharmaceutical preparations. The recoveries were from 95.1% to 103% and the relative standard deviations were in the range of 0.9% to 4.5%.     

Simultaneous quantitative and qualitative analysis of bioactive phenols in Dendrobium aurantiacum var. denneanum by high-performance liquid chromatography coupled with mass spectrometry and diode array detection

A novel high-performance liquid chromatographic method with mass spectrometry and diode array detection method for the simultaneous qualitative and quantitative analysis of bioactive phenols was developed. In total, nine chemically diverse phenols including five bibenzyls, three phenanthrenes and a coumarin were unambiguously identified in Dendrobium aurantiacum var. denneanum by comparison with the available references or reported data according to their retention behaviors, UV spectra and fragmentations of ESI-MS. The contents of the four main phenolic compounds, moscatilin, gigantol, moscatin and coumarin, in D. aurantiacum var. denneanum from the wild and various cultivated populations were determined by HPLC-UV. The sample preparation involved a rapid and simple procedure based on solid-phase extraction using a C(18) reversed-phase cartridge. The quantitative analysis was performed on a Beckman Coulter ODS column (5 microm, 250 x 4.6 mm) using a linear gradient elution system of acetonitrile-0.5% formic acid. The method was validated for linearity, limits of detection (LOD) and quantification (LOQ), precision and accuracy. Good results were obtained with respect to the overall intra- and inter-day variations (RSD less than 3.22%) and the percentage recoveries (ranging from 90.50 to 99.22%). Notable differences in the contents of phenols were observed among different cultivated populations. The samples colleted in April and May (spring), or October and November (autumn) accumulated much higher contents of phenols than those collected in other seasons.     

Simultaneous determination of water- and fat-soluble vitamins in pharmaceutical preparations by high-performance liquid chromatography coupled with diode array detection

Water- and fat-soluble vitamins were separated on a MetaChem Polaris C18-A (150 mm×4.6 mm, 3 μm particle size) in a single run using combined isocratic and linear gradient elution with a mobile phase consisting of 0.010% trifluoroacetic acid of pH 3.9 (solvent A) and methanol (solvent B) at the flow rate 0.7 ml min⁻¹. A linear gradient profile (A:B) started at 95:5 and was constant in the first 4 min, then linearly decreased up to 2:98 during the next 6 min, then it was constant in the next 20 min and finally linearly increased up to 95:5 ratio of water phase in the last 5 min of the separation. The most suitable detection wavelength for simultaneous vitamin determination was 280 nm. The method was applied for the solid sample of pharmaceutical preparation (B-Komplex), fortified powdered drinks (multi-vitamin) and food samples. The results were in good agreement with the declared values.     

Simultaneous analysis of nine active components in Gegen Qinlian preparations by high-performance liquid chromatography with diode array detection

HPLC with diode array detection (HPLC/DAD) was employed to determine the quantities of puerarin, daidzin, daidzein, berberine, palmatine, coptisine, baicalin, baicalein, and glycyrrhizin in Gegen Qinlian preparations of three different pharmaceutical forms including decoction, dispensing granule and pill. The calibration curves for the nine bioactive components were linear in the given concentration ranges. The precision of the method was in the range of 0.2 - 5.0% (RSD), and the recoveries of this method were between 96.5 and 104.1%. The proposed method was applicable to analyze Gegen Qinlian preparations.     

Simultaneous qualitation and quantification of four phytoecdysones in Radix Achyranthis Bidentatae by high-performance liquid chromatography with diode array detection

A high-performance liquid chromatographic method with diode array detection was developed and validated to simultaneously identify and quantify four phytoecdysones,i.e., polypodine B (1), ecdysterone (2), 25-R inokosterone (3) and 25-S inokosterone (4), in Radix Achyranthis Bidentatae. The analysis was performed using a C(18) column with 6% tetrahydrofuran aqueous and acetonitrile isocratic elution, and the detection was carried out by DAD at 242 nm. The identities of the analytes were determined by comparing the retention time and UV spectrum with those of reference compounds. The validation of the method included linearity, sensitivity, recovery and stability. All calibration curves of the four phytoecdysones showed good linear regression (r(2) >or= 0.9993). The limit of detection (S/N = 3) and limit of quantification (S/N = 10) were less than 7.5 and 12.3 ng, respectively. The method provided desirable repeatability with overall intra- and inter-day variations of less than 4.67%. The obtained recoveries varied between 95.1 and 104.4% while the relative standard deviations were below 4.85% (n = 3). The analysis results indicated that the contents of the investigated phytoecdysones in Radix Achyranthis Bidentatae from different locations were highly variant, and the genuine crude drug indigenous to Henan province did not possess the highest concentration of phytoecdysones.     

Simultaneous determination of thiols and disulfides by high-performance liquid chromatography with fluorescence detection

The proposed simultaneous determination of thiols and disulfides requires 4- (aminosulfonyl)- 7-fluor-2,1,3,-benzoxadiazole (ABD-F) as the pre-column derivatization reagent for thiols and ammonium 7-fluoro-2,1,3,-benzoxadiazole-4-sulfonate (SBD-F) for disulfides followed by chromatography. The thiols and disulfides in solution are treated with ABD-F at 60°C for 5 min in pH 9.3 borate buffer containing 1 nM disodium-EDTA. After removal of excess of ABD-F with ethyl acetate, the remaining disulfides in the aqueous phase are treated with SBD-F at 60°C for 20 min in the presence of tri-n-butylphosphine, a reducing agent. The ABD-thiols and SBD-thiols thus produced are separated by reversed-phase chromatography and detected by fluorimetry (380- nm excitation, 510 - nm emission). SBD-cystein, SBD-homocystein, ABD-homocysteine, ABD-cysteine, SBD-glu- tathione, ABD-homocystein, SBD-N-acetylcystein, ABD-glutatione and ABD-N-acetylcysteine are well separated by linear gradient elution from 0.15 M H₃PO₄/CH₃CN (96:4) to 0.15 M H₃PO₄/CH₃CN (85:15) over 30 min followed by isocratic elution with 0.15 M H₃PO₄/CH₃CN (85:15) fro 10 min. The detection limits for the derivatives are in the range 0.09–0.9 pmol. When the method was applied to the determination of thiols and disulfides in rat tissues, cystein (0.75 μmol g^-1) and cystine (0.62 μmol g^-1) were obtained in kidney and reduced glutathione (1.4–3.4 μmol g^-1) was observed in liver, spleen, heart and testicle.     

Simultaneous determination of six triazolic pesticide residues in apple and pear pulps by liquid chromatography with ultraviolet diode array detection

A method is described for the simultaneous determination of diclobutrazol, flusilazole, flutriafol, hexaconazole, paclobutrazol, and tetraconazole in apple and pear pulps used in baby food at a limit of 0.01 mg/kg. Apple and pear pulp samples are subjected to selective solid-phase microdispersion (SPMD) with SPE-ED Matrix-38 and acetone-cyclohexane, and the extracts are cleaned up on a Florisil cartridge with hexane-cyclohexane-acetone. The extracts are then analyzed by liquid chromatography with ultraviolet detection, using an octadecylsilane column with a gradient-programmed acetonitrile-water mobile phase. Recoveries were determined by spiking apple and pear pulps with the 6 pesticides under investigation at 0.1, 0.05, 0.03, and 0.01 mg/kg. Six determinations were performed at each level for each pesticide. Recoveries were > or = 70% at the 0.01 mg/kg level.     

Electrochemical treatment of textile dyes and their analysis by high-performance liquid chromatography with diode array detection

Several textile dyes were individually exposed to electrochemical treatment. Chromaticity variation and the formation of degradation products were followed using a UV spectrophotometer and HPLC with diode array detection. Dyes studied belong to the azo (color index, C.I. 15,510), methine (C.I. 48,013), indigo (C.I. 73,040), natural (C.I. 75,760) and arylmethane (C.I. 42,000) classes. Aliquots of the solutions treated at constant potential were analyzed and compared with control dye solutions. The final electrolysis solutions obtained by using different electrode materials: Pt, Ti and diamond presented different chromatograms. It was found that the novel (in this application) diamond electrode is efficient in studying the degradation of various dyes. Possible fragmentation and molecule moiety rearrangement are proposed as a result of the electrochemical treatment.     

Simultaneous determination of pharmaceutically active compounds in wastewater samples by solid phase extraction and high-performance liquid chromatography with diode array and fluorescence detectors

In the present work, an analytical method for the simultaneous determination of five anti-inflammatory drugs (acetaminophen, diclofenac, ibuprofen, ketoprofen and naproxen), an antiepileptic drug (carbamazepine) and a nervous stimulant (caffeine) is proposed for the routine analysis of these pharmaceuticals in wastewater influents and effluents from WWTPs. The method involves pre-concentration and clean-up by solid phase extraction (SPE) using Oasis HLB extraction cartridges. Final analysis of the selected pharmaceutical compounds was carried out by high-performance liquid chromatography (HPLC) with diode array detector (DAD). Confirmation of the presence of the fluorescence compounds (ibuprofen and naproxen) was performed by on-line fluorescence detection. Recoveries were ranged from 71 to 103% with relative standard deviation below 15.1%. Limits of quantification were in the range 6.2–319.8 and 3.0–160.0 ng ml⁻¹ for influent and effluent wastewater samples, respectively. The described method was applied to the determination of the drugs in wastewater samples from four treatment plants in Seville.     

Determination of melamine in pet food by enzyme immunoassay, high-performance liquid chromatography with diode array detection, and ultra-performance liquid chromatography with tandem mass spectrometry

Melamine in pet food (fortified or originally contaminated) was determined by enzyme immunoassay (EIA), high-performance liquid chromatography with diode array detection (HPLC-DAD), and ultra-performance liquid chromatography with tandem mass spectrometry (UPLC-MS/MS). The limits of detection (LOD) for EIA and HPLC-DAD were 0.02 and 0.1 microg/mL, respectively. The linear ranges of the calibration curves for EIA and HPLC-DAD were 0.02-0.5 and 0.1-500 microg/mL, respectively. The coefficient of determinations (r2) of the standard curves for EIA and HPLC were 0.9991 and 0.9999, respectively. Coefficient of variations from both inter- and intra-assay were <9.31%, and recovery range for all concentrations was between 71 and 105%. The r2 values between the EIA and HPLC-DAD methods for melamine analysis of the fortified and originally contaminated samples were 0.9973 and 0.9885. The r2 values for UPLC-MS/MS with HPLC-DAD and with EIA were 0.9566 and 0.9489, respectively.     

Simultaneous determination of twelve tea catechins by high-performance liquid chromatography with electrochemical detection.

A high-performance liquid chromatographic method with electrochemical detection was developed for the determination of twelve tea catechins including four major catechins: epicatechin (EC), epigallocatechin (EGC), epicatechin gallate (ECG) and epigallocatechin gallate (EGCG); four of their epimers at the C-2 position, C, GC, CG and GCG; and four methylated catechin derivatives, epigallocatechin-3-O-(3-O-methyl)gallate, gallocatechin-3-O-(3-O-methyl)gallate, epigallocatechin-3-O-(4-O-methyl)gallate and epicatechin-3-O-(3-O-methyl)gallate. These catechins were separated on an ODS C18 reversed-phase column by isocratic elution with 0.1 M NaH2PO4 buffer (pH 2.5)-acetonitrile (87:13) containing 0.1 mM EDTA.2Na. The detection limits (S/N = 3) of these catechins were approximately 10-40 pmol ml-1 at an applied voltage of 600 mV. Extracting these catechins from tea leaf powder with H2O-acetonitrile (1:1) at 30 degrees C for 40 min inhibited the epimerization at C-2 significantly from these epicatechins compared to extraction with hot water at 90 degrees C. This analytical method is sensitive to and appropriate for the simultaneous determination of various biologically active catechins in green tea.     

Determination of nifedipine in serum of women in preterm labor by high-performance liquid chromatography with diode array detection

Nifedipine (Nif) is widely used in treating cardiovascular disorders (especially hypertension) and for inhibiting preterm labor. A fully validated selective high-performance liquid chromatographic method with diode array detection, using solid-phase extraction, was developed for the determination of Nif in human serum. To assess specificity, Nif and its degradation products were separated on a Purospher RP-18 (5 microm, 125 x 4 mm) column plus a LiChrospher 100 RP-18 (5 microm, 4 x 4 mm) precolumn with a mobile phase of methanol-10 mM aqueous trifluoroacetic acid, pH 7.3 (57 + 43, v/v); chromatographic separation was followed by UV detection at 238 nm. For toxicological analysis, Nif in the presence of other calcium-channel antagonist drugs was identified under optimum chromatographic conditions. The calibration graph was constructed over the concentration range of 12.5-400 ng/mL in serum with good correlation (r = 0.9956). This method was not subject to interference by other plasma components and was successfully applied to the assay of Nif in spiked human serum and in serum of women in preterm labor after sublingual administration of 30 mg Nif per day divided into 3 equal doses. The mean recovery based on the ratio of the slopes of serum and mobile phase standard curves was 96.5%. The detection and quantification limits of the drug in spiked human serum were found to be 6 and 17.5 ng/mL, respectively. Validation of the method demonstrated good intraday and interday precision, which ranged from 2.18 to 6.67% and from 6.52 to 11.93%, respectively.     

Simultaneous determination of nitroimidazole residues in honey samples by high-performance liquid chromatography with ultraviolet detection

A rapid and sensitive high-performance liquid chromatography (LC) method was developed for the simultaneous determination of metronidazole (MNZ), dimetridazole (DMZ), ronidazole (RNZ), tinidazole (TNZ), and 2-hydroxymethyl-1-methyl-5-nitroimidazole (HMMNI) in honey. After extraction with ethyl acetate and evaporation, the residue containing the nitroimidazoles was dissolved in ethyl acetate-hexane and subjected to solid-phase extraction cleanup by amine extraction columns. The effluent was evaporated to dryness, and residues were dissolved and determined by LC with an ultraviolet detector set at 315 nm. The limits of detection were 1.0-2.0 ng/g for MNZ, DMZ, RNZ, TNZ, and HMMNI in honey. Average recoveries ranged from 71.5-101.4% in honey fortified at 10, 20, 50, and 100 ng/g. The method was validated for the analysis of real honey samples.     

Simultaneous determination of amiodarone and its metabolite desethylamiodarone by high-performance liquid chromatography with chemiluminescent detection

A novel method was developed for the determination of amiodarone and desethylamiodarone by high-performance liquid chromatography (HPLC) coupled with chemiluminescent (CL) detection. The procedure is based on the post-column photolysis of the analytes into photoproducts which are active in the tris(2,2′-bipyridyl)ruthenium(III) [Ru(bpy)₃³⁺] CL system. Ru(bpy)₃³⁺ was on-line generated by photo-oxidation of the Ru(II) complex in the presence of peroxydisulfate. The separation was carried out on a Mediterranea C₁₈ column with isocratic elution using a mixture of methanol and 0.017 mol L⁻¹ ammonium sulfate buffer of pH 6.8. Under the optimum conditions, analytical curves, based on standard solutions, were linear over the range 0.1-50 μg mL⁻¹ for amiodarone and 0.5-25 μg mL⁻¹ for desethylamiodarone. The detection limits of amiodarone and desethylamiodarone were 0.02 and 0.11 μg mL⁻¹, respectively. Intra- and inter-day precision values of 0.9% relative standard deviation (R.S.D.) (n = 10) and 1.6% R.S.D. (n = 15), respectively, were obtained. The method was applied successfully to the determination of these compounds in serum and pharmaceutical formulations.     

Simultaneous resolution of overlapping peaks in high-performance liquid chromatography and micellar electrokinetic chromatography with diode array detection using augmented iterative target transformation factor analysis

In this paper, augmentation has been applied to data matrices, which originate from hyphenated methods that share the same mode of detection, but use different separation methods, HPLC-DAD and MEKC-DAD. A novel method, wavelength shift eigenstructure tracking (WET), has been proposed for the alignment between the wavelength scale of both detectors. WET proves to be suitable for the detection as well as correction of wavelength shift between both detectors. After correction of the wavelength scale, data obtained on both systems have been augmented and submitted to iterative target transformation factor analysis. Augmented curve resolution provides significantly better estimates of the chromatographic and electrophoretic profiles and spectra than the use of non-augmented curve resolution on HPLC and MEKC data separately. It is particularly useful when the pure fraction of a chromatographic peak is less than 0.10. Finally, the relative weight of MEKC versus HPLC in augmentation may be increased using intensity and noise normalisation. However, since noise normalisation and its accompanying decrease in signal-to-noise ratio leads to a loss of information, and, since intensity normalisation may cause a failure of the augmented curve resolution algorithm, benefits and drawbacks of normalisation should be weighed on a case-by-case basis.