Ion chromatographic method for the determination of cations of group IA and IIA in water samples, pharmaceuticals and energy drinks by non-suppressed conductometric detection

An efficient ion chromatographic (IC) method was developed for the simultaneous quantitative determination of Li⁺, Na⁺, NH₄ ⁺, K⁺, Cs⁺, Ca²⁺, Mg²⁺, Sr²⁺, Ba²⁺ and Be²⁺ in energy drinks, pharmaceutical and drinking water samples by non-suppressed conductometric detection. The separation of ten cations including ammonium was achieved using a cation-exchange column and low conductivity mobile phase. The mobile phase consisted of tartaric acid, dipicolinic acid and boric acid. The separation of the cations was completed in less than 18 min, with a flow rate of 1.2 mL min⁻¹. The separation was not affected by the existence of cations Co²⁺, Cr³⁺, Cd²⁺, Cu²⁺, Bi³⁺, Ag⁺, Fe³⁺ and Zn²⁺ in concentrations up to 20 mg L⁻¹. Using an injection volume of 20 μL the obtained detection limits were 0.003 mg L⁻¹, 0.02 mg L⁻¹, 0.01 mg L⁻¹, 0.01 mg L⁻¹, 0.10 mg L⁻¹, 0.01 mg L⁻¹, 0.02 mg L⁻¹, 0.02 mg L⁻¹, 0.003 mg L⁻¹ and 0.1 mg L⁻¹, for Li⁺, Na⁺, NH⁴⁺, K⁺, Cs⁺, Ca²⁺, Mg²⁺, Sr²⁺, Be²⁺ and Ba²⁺ respectively. The intra-day repeatability (RSD%, n=5) ranged from 1.1% to 4.8%, and the inter-day (n=5) between 1.8% and 5.4% respectively. The method was applied to the analysis of various bottled and tap water, pharmaceutical preparations and energy drinks commercially available.      

Determination of water-soluble vitamins in pharmaceutical preparations by reversed-phase high-performance liquid chromatography with a mobile phase containing sodium dodecylsulphate andn-propanol

Summary A reversed-phase liquid chromatographic method with sodium dodecylsulphate-n-propanolwater as mobile phase has been used to separate and determine six water-soluble vitamins in twelve minutes. The analytical characteristics linear range, sensitivity, detection limits, and precision were evaluated. The lowest detection limits were those of nicotinic acid (not usually present in pharmaceutical products), 0.7 mgL⁻¹, nicotinamide, 1.3 mg L⁻¹, and pyridoxine, 1.4 mg L⁻¹. When the method was applied to the determination of the vitamins in pharmaceutical samples the values found agreed with those on the labels.     

LC Determination of Propylene Glycol in Human Plasma After Pre-Column Derivatization with Benzoyl Chloride

A simple high-performance liquid chromatographic method, using photodiode array detection was developed for the determination of propylene glycol in human plasma and in the fluid retreived after continuous veno-venous hemofiltration. The method entailed alkaline derivatization with benzoyl chloride and ethylene glycol as internal standard. The separation of the compounds, after extraction with pentane, was carried out on a Pursuit C8 column with UV-detection at 230 nm. Validation samples were analyzed with an accuracy between 95 and 105%, and intra- and inter-day coefficients of variation of less than 8%. The calibration curve was linear over a concentration range of 5–100 mg L⁻¹ with a detection limit of 1 mg L⁻¹. Blood plasma samples of several patients were analysed by using the prescribed method with propylene glycol concentrations varying from 5 to 98 mg L⁻¹. Compared to previously described LC methods, this method is ten times more sensitive and thus suitable for use in pharmacokinetic studies of propylene glycol.     

Monitoring of biologically active amines in cereals and cereal based food products by HPLC

Summary Biologically active amines (putreanine sulphate, N-acetyl putrescine, putrescine, cadaverine, histamine, agmatine, N-acetyl spermidine, spermidine, spermine) were separated and quantified in cereal flour and cereal products by a liquid chromatographic method. The method consists of the separation of ion pairs formed between biologically active amines and octanesulphonic acid on a reversed-phase column, postcolumn derivatization with o-phtalaldehyde-2-mercapthoethanol and spectrofluorometric detection. Results of the reliability study were satisfactory. The method was linear for each amine at 1–10 mg L⁻¹. Putrescine and spermidine were the only amines always detected in cereal flour and cereal products, ranging from 2.45 to 47.83 mg kg⁻¹ for putrescine and 3.27 to 37.14 mg kg⁻¹ for spermidine. The most important differences among types of samples were found in polyamine derivatives. Presented at: Balaton Symposium on High-Performance Separation Methods, Siófok, Hungary, September 3–5, 1997.     

Quantification of Teicoplanin in Human Plasma by Liquid Chromatography with Ultraviolet Detection

A simple high-performance liquid chromatographic method was developed for determining five major components of teicoplanin, designated A2–1, A2–2, A2–3, A2–4 and A2–5, in human plasma. Using piperacillin sodium as internal standard, teicoplanin in plasma samples was extracted by coextractive cleanup procedure. The extracts were injected into a Nova-Pak C₁₈ column maintained at ambient temperature. The mobile phase consisted of acetonitrile–0.1% trifluoroacetic acid (27:73, pH = 2.2), at a flow rate of 1.0 mL min⁻¹. The analytes were detected at the UV wavelength of 218 nm. The method was found to be linear over the concentration range of 2.5–50 mg L⁻¹ for teicoplanin (r = 0.9993 ± 0.0038), which covered the clinically expected trough plasma levels. The percentage error of the analytical method was below 9%. The intra- and inter-day reproducibility was adequate with coefficients of variation less than 7%. The chromatographic running time was 11 min. Thus, the method can be effectively applied to measure teicoplanin concentrations in clinical samples.     

Validated Enantioselective LC Method, with Precolumn Derivatization with Marfey’s Reagent, for Analysis of the Antiepileptic Drug Pregabalin in Bulk Drug Samples

An enantioselective high-performance liquid chromatographic method, with precolumn derivatization with Marfey’s chiral reagent, sodium 2,4-dinitro-5-fluorophenyl-l-alanine amide, has been developed for resolution of the enantiomers of a new antiepileptic drug, pregabalin, in the bulk drug. The diastereomers of the pregabalin enantiomers were resolved to baseline on a reversed-phase ODS column with a 60:40 (v/v) mixture of aqueous 0.2% triethylamine (pH adjusted to 3.5 with dilute orthophosphoric acid) and acetonitrile as mobile phase. Resolution between the diastereomers was not less than five. The method was extensively validated and proved to be robust. The calibration plot was indicative of an excellent linear relationship between response and concentration over the range 750 (LOQ) to 7,500 ng L⁻¹ for the R enantiomer. The limits of detection and quantification of the R enantiomer were 250 and 750 ng L⁻¹, respectively, for an injection volume of 10 μL. Recovery of the R enantiomer from bulk drug samples of pregabalin ranged from 97.5 to 101.76%. Solutions of pregabalin in water and in the mobile phase were found to be stable for at least 48 h. The method was found to be suitable and accurate for quantitative determination of the R enantiomer in the bulk drug. It can be also used to test the stability of samples of pregabalin.     

Evaluation of colon cancer elements contents in serum using statistical methods

Classification of normal and different cancer groups (TNM classification) with univariate and multivariate statistical methods according to the contents of Cu, Fe, Mn, Se, and Zn in blood serum is discussed. All serum samples were digested by acid mixture in a microwave mineralization unit prior to the analysis by atomic absorption spectrometry. Results show that univariate methods can distinguish normal and cancer groups. Level of selenium evaluated as arithmetic mean with its standard deviation in colorectal cancer patients was (42.61 ± 23.76) μg L⁻¹. Retransformed mean was used to evaluate levels of managanese (11.99 ± 1.71) μg L⁻¹, copper (1.05 ± 0.06) mg L⁻¹, zinc (2.14 ± 0.21) mg L⁻¹, and iron (1.82 ± 0.22) mg L⁻¹. Conclusions of multivariate statistical procedures (principal component analysis, hierarchical, and k-means clustering) do not correlate very well with the division of serum samples according to the TNM classification.     

A Validated Stability Indicating RPLC Method for Tazarotene

A simple, isocratic, rapid and accurate reversed phase high performance liquid chromatography method was developed for the quantitative determination of tazarotene. The developed method is also applicable for the related substance determination in bulk drugs. The chromatographic separation was achieved on a Hypersil C18 (250 mm × 4.6 mm 5 μm) column using water pH 2.5 with orthophosphoric acid:acetonitrile (15:85, v/v) as a mobile phase. The chromatographic resolutions between tazarotene and its potential impurity A and B were found greater than three. The limit of detection and limit of quantification of impurities were found to be 25 and 75 ng mL⁻¹. The percentage recovery of impurities in bulk drug sample was ranged from 96.8 to 103.5.The percentage recovery of tazarotene in bulk drug sample was ranged from 98.4 to 100.9. The developed RPLC method was validated with respect to linearity, accuracy, precision and robustness.     

Separation and determination of rutin and quercetin in the flowers ofSophora japonica L. by capillary electrophoresis with electrochemical detection

Summary Capillary electrophoresis with amperometric detection has been evaluated for the simultaneous determination of rutin and quercetin. The cyclic voltammogram, hydrodynamic voltammogram, and the effects of pH, concentration of buffer and sodium dodecyl sulfate (SDS), and amount of organic modifier on the separation and the detection were studied. The optimized conditions were: detection potential 1.2V, separation at 12 kV, 5 s at 15 kV for sample injection, running electrolyte 20 mmol L⁻¹ borate buffer, pH 8.8, containing 40 mmol L⁻¹ SDS and 10% acetonitrile. The detection limit of the method was low, 0.001 and 0.0005 mg mL⁻¹, for rutin and quercetin, respectively; the linear ranges were wide −0.005–0.5 and 0.005–0.4 mg mL⁻¹, respectively. The variations in peak current and migration time for eight consecutive injections of a standard solution containing 0.1 mg mL⁻¹ of each compound were 4.78 and 3.63%, and 6.50 and 2.59% for rutin and quercetin, respectively. The levels of the two compounds in traditional Chinese herbal drugs were easily determined.     

A Stability-Indicating LC Method for the Simultaneous Determination of Telmisartan and Ramipril in Dosage Form

A simple, rapid, and precise method is developed for the quantitative simultaneous estimation of telmisartan and ramipril in combined pharmaceutical dosage form. A chromatographic separation of the two drugs was achieved with an ACE 5 C₁₈, 25-cm analytical column using buffer–acetonitrile (55:45 v/v). The buffer used in mobile phase contains 0.1 M sodium perchlorate monohydrate in double distilled water pH adjusted 3.0 with trifluoroacetic acid. The instrumental settings are flow rate of 1.5 mL min⁻¹, column temperature at 30 °C, and detector wavelength of 215 nm using a photodiode array detector. The resolution between ramipril and telmisartan were found to be more than 5. Theoretical plates for ramipril and telmisartan were 13,022 and 6,629. Tailing factor for ramipril and telmisartan was 0.94 and 0.98. Telmisartan, ramipril and their combination drug product were exposed to thermal, photolytic, hydrolytic and oxidative stress conditions, and the stressed samples were analysed by the proposed method. Peak homogeneity data of telmisartan and ramipril is obtained using photodiode array detector, in the stressed sample chromatograms, demonstrated the specificity of the method for their estimation in presence of degradants. The described method shows excellent linearity over a range of 20–400 μg mL⁻¹ for telmisartan and 2.5–50 μg mL⁻¹ for ramipril. The correlation coefficient for telmisartan and ramipril are 1. The relative standard deviation for six measurements in two sets of each drug in tablets was always less than 2%. The proposed method was found to be suitable and accurate for quantitative determination and the stability study of telmisartan and ramipril in pharmaceutical preparations.     

Determination of galacturonic acid content in pectin from fruit juices by liquid chromatographydiode array detection-electrospray ionization tandem mass spectrometry

A reversed-phase high-performance liquid chromatographic (HPLC) method is applied for the determination of galacturonic acid (GA) of pectins in different commercial fruit juices. The separation was carried out on a C₁₈ column using precolumn derivatization with p-aminobenzoic acid (p-ABA) and UV detection at 304 nm. The identification of GA was confirmed by high-performance liquid chromatography/electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) in positive ion mode. The concentration of GA in the samples analyzed ranged from 12.9 ± 0.5 to 49.4 ± 0.5 mg_GA L⁻¹. Amongst the samples analyzed, mango juice was found to be richest in GA content, and therefore a good source of pectins. Detection and quantification limits of the described methodology were 1.2 and 3.9 mg L⁻¹, respectively. Quantitative GA recoveries in the beverages had a range between 90 and 98%. The results showed that the HPLC method proposed was precise and suitable for the identification and quantification of GA in commercial fruit juices.     

Quantitative analysis of nicotinic acid, nicotinamide and 3-cyanopyridine in industrial effluent by high performance liquid chromatography

A rapid and reliable high performance liquid chromatographic (HPLC) method for the simultaneous determination of heterocyclic compounds, namely nicotinic acid, nicotinamide, and 3-cyanopyridine, in industrial effluent is described. A Φ4.6 mm × 150 mm, 5 μm C-18 reversed phase stationery phase, and a methanol-acetonitrile-water tertiary mobile phase (20:20:60 v/v) were used for separation. The detection wavelength of a diode array (DAD) was set at 216 nm with a bandwidth of 16 nm. Phenol was used as an internal standard. The regression equations revealed a linear relationship between the concentration of the analytes injected and the peak area detected by DAD. The limits of detection (S/N = 3) ranged from 0.70 to 1.18 mg L⁻¹, the recoveries ranged from 87% to 102% and the precision expressed as % RSD intra-day and inter-day varied from 0.9 to 3.9 and 1.2 to 5.6, respectively. This method is rapid, sensitive and suitable for the monitoring of nicotinic acid, nicotinamide, and 3-cyanopyridine in effluent of related pharmaceutical manufacturing plants.     

Direct detection of nitric oxide in human blood serum by use of 1,3,5,7-tetramethyl-8-(3′,4′-diaminophenyl)difluoroboradiaza-s-indacene with HPLC

To study the relationship between amounts of nitric oxide (NO) in blood and the development of ischemic cardio-cerebrovascular diseases, trace NO in human blood serum has, for the first time, been determined by use of a 1,3,5,7-tetramethyl-8-(3′,4′-diaminophenyl)difluoroboradiaza-s-indacene (TMDABODIPY)-based HPLC method. The proposed method is simple, rapid, and efficient, owing to its high sensitivity and good selectivity for NO. TMDABODIPY and its NO derivative are separated to baseline in 4 min, with simple separation conditions, on a C₁₈ column eluted with 50 mmol L⁻¹ ethanolamine in methanol. The derivative is detected by fluorescence at an emission wavelength of 507 nm with excitation at 498 nm. The response is a linear function of concentration in the range 0.8–800 nmol L⁻¹ NO. The detection limit can reach 2×10⁻¹¹ mol L⁻¹ (signal-to-noise ratio=3). The method has been used to detect NO in the serum of patients with five kinds of ischemic cardio-cerebrovascular disease and two diseases closely connected with ischemic cardio-cerebrovascular diseases. Recoveries of NO from spiked serum samples were between 96.58 and 105.71% and concentrations of NO observed in real samples were at 10⁻⁷ mol L⁻¹ levels. Our studies indicate that the proposed TMDABODIPY-based HPLC technique can be developed into a sensitive and new method for clinical assay and pathology research.     

Sensitive determination of nitrogenous hydrochloride drugs via their reaction with ammonium molybdate

In a hydrochloric acid medium, benzhexol hydrochloride, cyproheptadine hydrochloride, and maprotiline hydrochloride, can react with ammonium molybdate to form ion-association complexes by virtue of electrostatic attraction and hydrophobic interaction which result in a significant enhancement of the resonance light scattering intensity. The maximum scattering wavelengths were found at 364 nm, 364 nm, and 381 nm for benzhexol hydrochloride, cyproheptadine hydrochloride, and maprotiline hydrochloride systems, respectively. Spectral characteristics of the three systems, influencing factors, and optimum conditions were investigated. The reason of resonance light-scattering enhancement and the mechanism of interactions between the drugs and ammonium molybdate were also discussed. Based on the linear relationship between the enhanced intensity of resonance light scattering and the concentration of drugs, a highly sensitive method for the determination of the three drugs was developed, the detection limits being 0.0110 μmol L⁻¹, 0.0038 μmol L⁻¹, and 0.0155 μmol L⁻¹ for benzhexol hydrochloride, cyproheptadine hydrochloride, and maprotiline hydrochloride, respectively. The method was successfully applied to the determination of the investigated drugs in pharmaceutical, serum, and urine samples.     

Speciation analysis of inorganic antimony in soil using HPLC-ID-ICP-MS

Speciation analysis of Sb(III) and Sb(V) in a soil sample was performed through extraction and on-line isotope dilution concentration determination after a chromatographic separation. The total Sb concentration found in a through traffic contaminated soil sample was (4.17 μg g⁻¹, 0.3 μg g⁻¹ SD, n=6). It was determined using ICP-MS after soil digestion using the sodium peroxide sintering method. The optimized extraction procedure for speciation analysis was carried out using 100 mmol L⁻¹ citric acid at pH 2.08 by applying an ultrasonic bath for 45 min at room temperature. The effects of citric acid concentration (0–500 mmol L⁻¹), pH (1–6), and temperature (30–60°C) on inorganic antimony species distribution in the examined sample were studied and optimized. The separation of Sb(III) and Sb(V) was achieved using an anion exchange column (PRP-X100) and 10 mmol L⁻¹ EDTA and 1 mmol L⁻¹ phthalic acid at pH 4.5 as a mobile phase. The eluent from the HPLC was mixed with an enriched (94.2%) ¹²³Sb spike solution that was pumped by a peristaltic pump with a constant flow rate (0.5 mL min⁻¹) in a three-way valve. The blend passed directly to the Conikal nebulizer of the ICP-MS. By using the above extraction procedure and methodology, 43.2% Sb(V) (2.9% RSD, n=3) and 6.0% Sb(III) (1.3% RSD, n=3) of total Sb found in the sample could be detected. The detection limits achieved by the proposed method were 20 ng L⁻¹ and 65 ng L⁻¹ for Sb(V) and Sb(III), respectively. The precision, evaluated by using RSD with 100 ng L⁻¹ calibration solutions, was 2.7% and 3.2% (n=6) for Sb(V) and Sb(III), respectively, in aqueous solutions.     

Application of an in situ plated lead film electrode to the analysis of testosterone by adsorptive stripping voltammetry

Adsorptive stripping voltammetry is a very sensitive and selective method for determination of drugs and organic substances in biological fluids. We have shown that determination of testosterone by adsorptive stripping voltammetry is possible using a lead film electrode. The lead film plating process and accumulation of testosterone were performed simultaneously from an acetate buffer solution of pH = 5.2 at a potential of −1.1 V. The measurements were carried out in undeaerated solutions. The detection limit was 9 × 10⁻⁹ mol L⁻¹ for an accumulation time of 120 s; the relative standard deviation for 1 × 10⁻⁷ mol L⁻¹ testosterone was 3.8%. The proposed voltammetric procedure for determination of testosterone could be applied to its determination in a pharmaceutical preparation and human urine samples directly without any separation steps.     

Applicability of a liquid membrane in enrichment and determination of nickel traces from natural waters

In this work, a bulk liquid membrane method has been applied for Ni enrichment and separation from natural waters. The carrier-mediated transport was accomplished by pyridine-2-acetaldehyde benzoylhydrazone dissolved in toluene as a complexing agent. The preconcentration was achieved through pH control of source and receiving solutions via a counterflow of protons. The main variables were optimized by using a modified simplex technique. High transport efficiencies (101.2 ± 1.8–99.7 ± 4.2%) were provided by the carrier for nickel ions in a receiving phase of 0.31 mol L⁻¹ nitric acid after 9–13 h depending on sample salinity. The precision of the method was 2.05% (without a saline matrix) and 4.04% (with 40 g L⁻¹ NaCl) at the 95% confidence level and the detection limit of the blank was 0.015 μg L⁻¹ Ni for detection by atomic absorption spectroscopy. The applicability of the method was tested on certified reference and real water samples with successful results, even for saline samples. The relative errors were −0.60% for certified reference materials and ranged from −0.39 to 2.90% and from 0.3 to 11.05% for real samples, obtained by comparison of inductively coupled plasma mass spectrometry and adsorptive cathodic stripping voltammetry measurements, respectively.     

A Validated Chiral LC Method for the Enantiomeric Separation of Repaglinide on Amylose Based Stationary Phase

A simple, isocratic, normal phase chiral HPLC method was developed and validated for the enantiomeric separation of repaglinide, (S)-(+)-2-ethoxy-4-N [1-(2-piperidinophenyl)-3-methyl-1-butyl] aminocarbonylmethyl] benzoic acid, an antidiabetic in bulk drug substance. The enantiomers of repaglinide were resolved on a ChiralPak AD-H (amylose based stationary phase) column using a mobile phase consisting of n-hexane: 2-propanol:trifluoroacetic acid (95:5:0.2 v/v/v) at a flow rate of 1.0 mL min⁻¹. The resolution between the enantiomers was found to be not >3.5 in optimized method. The presence of trifluoroacetic acid in the mobile phase played an important role, in enhancing chromatographic efficiency and resolution between the enantiomers. The developed method was extensively validated and proved to be robust. The calibration curve for (R)-enantiomer showed excellent linearity over the concentration range of 900 ng mL⁻¹ (LOQ) to 6,000 ng mL⁻¹. The limit of detection and limit of quantification for (R)-enantiomer were 300 and 900 ng mL⁻¹, respectively. The percentage recovery of the (R)-enantiomer ranged between 98.3 and 101.05% in bulk drug samples of repaglinide. Repaglinide sample solution and mobile phase were found to be stable up to 48 h. The developed method was found to be enantioselective, accurate, precise and suitable for quantitative determination of (R)-enantiomer in bulk drug substance.     

Chiral separation of N-(trans-4-isopropylcyclohexylcarbonyl)-D,L-phenylalanine isomers by high performance liquid chromatography

Summary A high-performance liquid chromatographic method was developed for the chiral separation of a new anti-diabetic agent, N-(trans-4-isopropylcyclohexylcarbonyl)-D-phenylalanine, and its L-enantiomer. The separation was performed on a Sumichiral OA-3300 column. Optimized mobile phase was 0.025 mol.L⁻¹ ammonium acetate in methanol solution. UV detection was at 210 nm. Baseline chiral separation was obtained within 12 minutes. The detection limits are 80 pg for the D-enantiomer and 120 pg for the L-enantiomer. RSD of the method was below 1% (n=5).     

A Capillary GC Method Using Nitrogen Phosphorus Detection for Determination of Topiramate in Patients with Epilepsy

A new capillary gas chromatographic assay with nitrogen phosphorus detection for the determination of topiramate in the serum was developed and validated. The sample procedure included a liquid–liquid extraction of 0.1 mL of alkalized sample with ethyl acetate. The organic solvent was evaporated, and flash methylation of topiramate and internal standard 5-(p-methylphenyl)-5-phenylhydantoin with trimethylanilinium hydroxide (0.07 mol L⁻¹ in methanol) was performed. The structure of derivatization product was confirmed using gas chromatography–mass spectrometry. Validation experiments certified suitability of bioanalytical method in the monitoring of serum concentrations in epileptic patients. The limit of detection was found to be 1.69 μmol L⁻¹. The range of applicability and linearity was established from 4.35 to 69.62 μmol L⁻¹. The accuracy and precision reached acceptable values from 86.15 to 109.5% (recovery) respectively, values were from 2.19 to 11.03% (RSD). No interference was found from endogenous substances or studied antiepileptic drugs.