Development and Validation of a Chiral Liquid Chromatographic Method for Quantitative Determination of (+)-OTX015 in (−)-OTX015

A simple, rapid and robust enantioselective method was developed and validated for the quantitation of OTX015 enantiomers [(−)-OTX015 and (+)-OTX015] with ultrahigh-performance liquid chromatography (UHPLC) as per ICH guidelines. The active [(−)-OTX015] and inactive [(+)-OTX015] enantiomers were resolved on a Chiralpak-IA column using methanol consisting of 0.1 % diethyl amine at a flow rate of 1.0 mL min⁻¹. The resolution between the enantiomers was found to be more than 3.7 in the optimized method. The developed method was extensively validated and proven to be robust. The calibration curve for (+)-OTX015 showed excellent linearity over the concentration range of 10–100 µg mL⁻¹. The limit of detection and the limit on quantitation for (+)-OTX015 were 5 and 10 µg mL⁻¹, respectively. The recovery for (+)-OTX015 ranged between 100.7 and 102.5 % in the bulk drug sample of (−)-OTX015. The proposed method was found to be suitable and accurate for quantitative determination of (+)-OTX015 in bulk drug.

     

Study on the Enantioselective Degradation of Imazethapyr in Soil by CE

A method was developed for the separation of imazethapyr enantiomers using capillary electrophoresis and large volume injection. Optimized buffer conditions were found using 6% hydroxypropyl-β-cyclodextrin as the chiral selector at pH 11 with an injection time of 20 s (0.5 psi). The limits of detection (LOD, S/N = 3) were 0.22 and 0.23 mg L⁻¹ for the two imazethapyr enantiomers (imazethapyr-I and imazethapyr-II) respectively. The relative standard deviation was less than 5%. This method was successfully used in the study of enantioselective degradation of this herbicide in field soil and the results suggested that imazethapyr-I degraded at a higher rate when compared with imazethapyr-II.

     

Determination of Racemic Ketorolac,Ketorolac Enantiomers and Their Metabolites in Human Plasma and Urine by LC–UV,Applied in Clinical Study During and After Pregnancy

In order to enhance the sensitivity and to develop a faster direct method for plasma and urine quantification of racemic ketorolac, its metabolites (p-hydroxy-ketorolac and ketorolac glucuronides) and ketorolac enantiomers, we developed an extraction procedure based on solid-phase extraction combined with specific and fast chromatographic separation. Extraction and chromatography resulted in cleaner chromatograms without interfering compounds. In both plasma and urine, linearity of the standard curves for racemic ketorolac and p-hydroxy-ketorolac was validated in the concentration range 0.025–10 mg L⁻¹, while for ketorolac enantiomers in the concentration range 0.025–5 mg L⁻¹. The lower limit of quantification was two times lower than in earlier described methods. The developed method was suitable for direct quantification of racemic ketorolac, p-hydroxy-ketorolac and ketorolac enantiomers in plasma and urine samples in women at delivery and in postpartum, enabling us to document significant intra-individual differences in pharmacokinetics between these physiological states.

     

Nonderivatized Sarcosine Analysis by Gas Chromatography after Solid-Phase Microextraction by Newly Synthesized Monolithic Molecularly Imprinted Polymer

In this study, a simple method for analysis of nonderivatized sarcosine was developed by gas chromatography. It is based on solid-phase microextraction of sarcosine on a novel synthesized solid-phase microextraction (SPME) fiber. A monolithic SPME fiber was fabricated based on a molecularly imprinted polymer that could be coupled with gas chromatography for extraction and determination of sarcosine. Extraction time, pH, and ionic strength were investigated as important factors in the extraction procedure. The fabricated fiber was firm, inexpensive, stable, and selective, which are vital characteristics for SPME. The selectivity of the fabricated fiber in relation to analog compounds was also investigated. Under optimum conditions, the calibration curve was linear in the range of 1–100 mg L⁻¹ (R ² = 0.987). High extraction efficiency for sarcosine was obtained with a detection limit of 0.37 mg L⁻¹. The fabricated fiber was successfully applied for SPME of sarcosine from urine after its extraction, followed by gas chromatography flame ionization detector analysis.

     

Determination of Repertaxin Enantiomeric Purity by HPLC on Chiral Stationary Phase

A precise and sensitive LC method for the determination of repertaxin enantiomeric purity has been developed and validated. Baseline separation with a resolution higher than 2.0 was accomplished within 20 min using a Chiralpak AD-H column (250 × 4.6 mm; particle size 5 μm) and n-hexane:2-propanol (90:10 v/v) as mobile phase at a flow rate of 1 mL min⁻¹. Eluted analytes were monitored by UV detection at 260 nm. The effects of mobile phase composition, temperature and flow rate on enantiomeric selectivity and on resolution of enantiomers were investigated. Calibration curves were plotted within the concentration range between 0.002 and 1.0 mg mL⁻¹ (n = 3), and relative standard deviation (RSD) of the inter-batch assay and intra-batch assay was less than 1.27 and 1.16 %. LOD and LOQ for repertaxin were 0.65 and 2.19 μg mL⁻¹; those for its enantiomer were 0.70 and 2.34 μg mL⁻¹, respectively. The method was evaluated and validated by analysis of bulk samples of repertaxin of different enantiomeric purity. It was demonstrated that the method was accurate, robust, and sensitive, and enabled practical analysis of real samples.

     

Stability-Indicating Liquid Chromatography–Spectrophotometric UV Method for the Simultaneous Determination of Marbofloxacin,Dexamethasone and Clotrimazole in a Liquid Pharmaceutical Dosage Form

A novel, simple and reliable reversed-phase liquid chromatography (LC)–spectrophotometric UV stability-indicating method was developed and validated for the simultaneous assay of marbofloxacin, clotrimazole and dexamethasone acetate in the presence of their impurities and degradation products in a pharmaceutical formulation for veterinary use. A C18 (75 × 4.6 mm, 4 µm) column was used with an acetonitrile–ammonium acetate mixture as mobile phase delivered with gradient elution. A diode-array detection was used in the 200–400 nm range and the detection wavelength was set at 260 nm. Validation carried out on the pharmaceutical dosage form, according to Veterinary International Conference on Harmonization guidelines, demonstrated excellent specificity, linearity, precision, accuracy and robustness. Excellent specificity with respect to vehicle and degradation products obtained after forced degradation (i.e., oxidation, acid, alkaline and thermal degradation) was demonstrated. As for linearity, the LC–UV assay method is applicable in the 0.180–0.420 mg mL⁻¹ concentration range for marbofloxacin (r ² = 0.99), 0.060–0.140 mg mL⁻¹ for dexamethasone acetate (r ² = 0.97) and 0.600–1.400 mg mL⁻¹ for clotrimazole (r ² = 0.98). Very good repeatability (RSD < 0.8 %) and inter-day precision (RSD < 2.5 %) were observed for all analytes. Accuracy was in the 93–104 %, 98–111 % and 99–108 % confidence interval (95 %) for marbofloxacin, dexamethasone acetate and clotrimazole, respectively. The variations (±20 %) of mobile phase flow rate and pH, and oven column temperature did not exhibit an impact on the analyte content accuracy, demonstrating the robustness of the method. The LC–UV method here developed and validated may be used routinely for quality control.

     

Determination of the Enantiomeric Purity of Trelagliptin by Pre-Column Derivatization and Liquid Chromatography on a Chiral Stationary Phase

A precise and sensitive LC method for determination of enantiomeric purity of trelagliptin has been developed and validated. Pre-column derivatization was performed before separation. Baseline separation with a resolution factor >2.5 was accomplished within 10 min by use of a Chiralpak AD column (250 × 4.6 mm; particle size 5 µm) and n-hexane–2-propanol (90:10 v/v) as mobile phase at a flow rate of 1 mL min⁻¹. Eluted analytes were monitored by UV detection at 260 nm. The effects of mobile phase composition and temperature on enantiomeric selectivity and on resolution of enantiomers were thoroughly investigated. Calibration curves were plotted within the concentration range 0.005–2 mg mL⁻¹ (n = 12), and recoveries between 98.23 and 101.34 % were obtained, with relative standard deviation (RSD) <1.39 %. LOD and LOQ for the trelagliptin derivative were 1.51 and 5.03 µg mL⁻¹; those for its enantiomer were 1.49 and 4.94 µg mL⁻¹, respectively. The method was evaluated and validated by analysis of bulk samples of trelagliptin of different enantiomeric purity. It was demonstrated that the method was accurate, robust, and sensitive, and enabled practical analysis of real samples.

     

Assessment of a Plantibody HB-01 Purification Strategy at Different Scales

Transgenic plant investigations focus on fine-regulation of the recombinant protein expression and purification strategies. In this article, preliminary experiments were done at analytical-scale to decide the best plantibody HB-01 purification strategy. Once it was assumed, the purification efficiency was assessed at different scales (10–600 kg of biomass). The plantibody purity measured by SDS-PAGE and LC-GF was over 90%, yielding 9.9 ± 6.2–18.6 ± 0.9 mg plantibody kg⁻¹ of biomass and 39.9 ± 7.9–48.7 ± 2.1% of recovery. Significant differences were not observed among these parameters at these scales. Plant DNA contents were <3.3 ng mg⁻¹ of plantibody, which is considered very low for the plantibody HB-01 application in the hepatitis B vaccine production.

     

Determination of Picoxystrobin and Pyraclostrobin by MEKC with On-Line Analyte Concentration

Micellar electrokinetic capillary chromatography was used for the determination of picoxystrobin and pyraclostrobin. The background electrolyte consisted of borate buffer (40 mmol L⁻¹ pH 8.5), SDS (30 mmol L⁻¹) and acetonitrile (15% in volume). Runs were made at 25 °C with 25 kV applied potential. The developed method was applied to analyte fortified urine samples. On-line analyte concentration, combined with a capillary of a longer optical path length, allowed limits of quantification of 8.6 × 10⁻⁸ mol L⁻¹ for picoxystrobin and 1.8 × 10⁻⁷ mol L⁻¹ for pyraclostrobin.

     

Residue Analysis of Propionylbrassinolide in Fruit and Vegetables by GC–MS

A rapid and simple method for the determination of propionylbrassinolide residues in tomatoes, apples and grapes using GC–MS is reported. Samples were extracted with acetonitrile, and the extracts were analyzed without any further clean-up. The results showed good linearity (r ² > 0.99) with standard solutions over the concentration range of 0.5–50 mg L⁻¹. The LODs and LOQs of propionylbrassinolide were 0.15 and 0.5 mg kg⁻¹ in all samples. Recoveries were in the range of 81.9–111.2%, with corresponding RSDs of 4.6–12.9% for three fortified levels. Intra- and inter-day RSDs were in the ranges of 1.5–14.2% and 5.3–15.6%. It was demonstrated that the proposed method is simple and efficient, and particularly suitable for detecting propionylbrassinolide residues in fruit and vegetables.

     

Modified carbon nanotubes as a sorbent for solid-phase extraction of gold,and its determination by graphite furnace atomic absorption spectrometry

A simple, sensitive and accurate method was developed for solid-phase extraction and preconcentration of trace levels of gold in various samples. It is based on the adsorption of gold on modified oxidized multi-walled carbon nanotubes prior to its determination by graphite furnace atomic absorption spectrometry. The type and volume of eluent solution, sample pH value, flow rates of sample and eluent, sorption capacity and breakthrough volume were optimized. Under these conditions, the method showed linearity in the range of 0.2–6.0 ng L⁻¹ with coefficients of determination of >0.99 in the sample. The relative standard deviation for seven replicate determinations of gold (at a level of 0.6 ng L⁻¹) is ±3.8 %, the detection limit is 31 pg L⁻¹ (in the initial solution and at an S/N ratio of 3; for n = 8), and the enrichment factor is 200. The sorption capacity of the modified MWCNTs for gold(III) is 4.15 mg g⁻¹. The procedure was successfully applied to the determination of gold in (spiked) water samples, human hair, human urine and standard reference material with recoveries ranging from 97.0 to 104.2 %.

A sorbent based on modified carbon nanotubes was prepared and used to extract gold ion from various samples prior to its determination by graphite furnace atomic absorption spectrometry

     

Fast Determination of Phenolic Compounds in Brazilian Wines from Vale do São Francisco Region by CE

Six phenolic compounds were separated and determined by capillary zone electrophoresis in red wine from Brazil’s region Vale do São Francisco with total analysis time of 12 min. The limit of detections varied from 1.59 to 2.24 mg L^?1. The relative standard deviations (for n = 6) varied from 0.28 to 3.50 %. The red wine samples analyzed were bought in the local market and the phenolic compound recoveries were in the range of 98–101 %. The concentrations of gallic acid in the samples of wines varied from 16.0 to 42.0 mg L^?1, caffeic acid (3.16–5.18 mg L^?1), syringic acid (5.73–13.0 mg L^?1), kaempferol (2.32–4.33 mg L^?1), quercetin (1.68–4.03 mg L^?1), myricetin (7.52–25.1 mg L^?1). The concentrations found agree with data reported in the literature.     

Sequential Determination of Inorganic Cations and Anions in Cerebrospinal Fluid by Microchip Electrophoresis

Analysis of inorganic ions in cerebrospinal fluid (CSF) is used mainly in the diagnostics of central nervous system diseases, such as Alzheimer’s disease or multiple sclerosis. A new analytical method for fast determination of inorganic cations (ammonium, calcium, magnesium, sodium and potassium) and anions (chloride, sulfate, nitrite and nitrate) in CSF on an electrophoretic microchip was developed in this context. Zone electrophoresis (ZE) separations were performed on the microchip with coupled channels (CC) and contact conductivity detection. Two different propionate background electrolytes were used for the sequential determination of cations at pH 3.1 and anions at pH 4.3. ZE was used for the determination of cationic constituents while ZE–ZE approach was employed for the determination of chloride in the first separation channel on the CC microchip and other anionic micro-constituents in the second channel. LOD values were in the range of 0.003–0.012 mg L⁻¹ and 0.019–0.047 mg L⁻¹ for cations and anions, respectively. Repeatability of migration time was up to 1.2 % for both cations and anions. Repeatability of peak area ranged from 0.3 to 5.6 % for cations and from 0.6 to 6.0 % for anions. Recovery of both cations and anions was in the range 90–106 %. CSF samples were only diluted appropriately without other sample pretreatment prior to analysis. Developed sequential method is suitable for fast determination of the studied cations and anions in CSF with total analysis time <15 min.

     

Fluorimetric Determination of p -Hydroxybenzoic Acid in Beer as α -Cyclodextrin Inclusion Complex

Abstract

The inclusion complex of p-hydroxyenzoic acid and α- and β-cyclodextrin has been studied by fluorescence spectroscopy. To describe quantitatively complex formation between α-cyclodextrin (α-CD) and p-hydroxybenzoic acid, an association constant of 967 ± 14 M^?1 at 21°C was obtained. The inclusion complex has been used to determine p-hydroxybenzoic acid in the range 0.15–1.00 mg L^?1 (RSD 4.5%, n = 8). Application of the method to determination of p-hydroxybenzoic acid in beer samples gave an endogenous content of 1.25 mg L^?1.     

GC–MS Method Development for the Analyses of Thiophenes from Solvent Extracts of Tagetes patula L

A method for isotachophoretic determination of potassium and ammonium cations in fertilizers and silage was developed. A capillary of 0.4 mm i.d. and 100 mm effective length made of fluorinated ethylene–propylene copolymer was filled with an electrolyte system consisting of 10 mmol L⁻¹ RbOH + 0.1% (w/v) hydroxyethylcellulose, adjusted to pH 9.0 with l-histidine (leading electrolyte) and 10 mmol L⁻¹ lithium citrate (terminating electrolyte). Using contactless conductivity detection, the calibration curves in the tested concentration range up to 0.5 mmol L⁻¹ were linear for both cations. The concentration detection limits for potassium and ammonium were 2.9 and 2.7 μmol L⁻¹, respectively. RSD values of step lengths (n = 6) were 1.3% for potassium and 1.5% for ammonium. The separation time was about 20 min. Similar results were obtained with cesium cation used as the leading ion, however, in the system with rubidium better resolution of other cations present in tested matrices was reached. The elaborated method is simple to perform, sufficiently sensitive and accurate and can be recommended as an alternative procedure to the methods used so far for the determination of potassium and ammonium.

     

Rapid Determination of Trifluoromethanesulfonate and p-Toluenesulfonate by Ion-Pair Chromatography Using a Reversed-Phase Silica-Based Monolithic Column: Application to the Analysis of Ionic Liquids

A method of ion-pair chromatography was developed on a reversed-phase silica-based monolithic column for the fast and simultaneous determination of trifluoromethanesulfonate (CF₃SO₃ ⁻) and p-toluenesulfonate (C₇H₇SO₃ ⁻). The analysis was performed using a mobile phase of tetrabutylammonium hydroxide + citric acid + acetonitrile on the Chromolith Speed ROD RP-18e column with direct conductivity detection. The effects of the eluent, column temperature and flow rate on the retention of the anions were investigated. The experimental phenomenon was discussed according to hydrophobic interaction and ion-exchange mechanism in the separation. The optimized chromatographic conditions were selected. The optimized eluent for the separation consisted of 0.2 mmol L⁻¹ tetrabutylammonium hydroxide + 0.10 mmol L⁻¹ citric acid + 9% acetonitrile (pH 5.5). The flow rate was set at 6.0 mL min⁻¹. The column temperature was 25 °C. Under the optimal conditions, the better separation of CF₃SO₃ ⁻ and C₇H₇SO₃ ⁻ was achieved without any interference by other anions (Cl⁻, Br⁻, I⁻, NO₃ ⁻, SO₄ ²⁻, ClO₃ ⁻, BF₄ ⁻ and PF₆ ⁻). The detection limit (S/N = 3) was 0.28 and 0.71 mg L⁻¹ for CF₃SO₃ ⁻ and C₇H₇SO₃ ⁻, respectively. The method has been applied to the determination of CF₃SO₃ ⁻ and C₇H₇SO₃ ⁻ in ionic liquids. The spiked recoveries of CF₃SO₃ ⁻ and C₇H₇SO₃ ⁻ were 101.1 and 100.2%, respectively.

     

Determination of Spectinomycin in Human Urine Using CE Coupled with Electrogenerated Chemiluminescence

A method of capillary electrophoresis with electrogenerated chemiluminescence was developed for the detection of spectinomycin. The linear ranges were from 0.01 to 1.0 mg mL⁻¹ for spectinomycin. The limit of detection for spectinomycin with a signal-to-noise ratio of 3:1 was found to be 4.0 µg mL⁻¹. For practical application perchloric acid was used to eliminate the protein contained in human urine, which is very harmful to electrophoresis separation. The recoveries of spectinomycin at different levels in human urine were between 91.1 and 106.5%. The RSD was between 2.6 and 4.8% (n = 5–6). It was valuable in clinical and biochemical laboratories for monitoring the drug for various purposes.

     

CE, with Hydroxypropyl-β-Cyclodextrin as Chiral Selector, for Separation and Determination of the Enantiomers of Amlodipine in the Serum of Hypertension Patients

A capillary electrophoretic method for separation of the enantiomers of amlodipine in the serum of hypertension patients has been established and validated. The two enantiomers were separated in a fused-silica capillary with phosphate running buffer (75 mmol L^?1, pH 2.5) containing 15 mmol L^?1 hydroxypropyl-β-cyclodextrin (HP-β-CD). The effects on the separation of buffer pH and concentration, separation potential, and concentration of HP-β-CD were investigated. The range of quantitation for both enantiomers was 2.0–16.0 μg mL^?1. Intra-day and inter-day relative standard deviation (RSD; n = 5) was <10%. The limits of detection (LOD) and quantification (LOQ) of the amlodipine enantiomers, at 214 nm, were approximately 0.5 and 0.7 μg mL^?1, respectively (S/N = 3 and 10, respectively; 5-s injection). Recovery was always >85%. Results from enantiomer separation and quantification showed that concentrations of the enantiomers of amlodipine in serum from an elderly patient were higher than in serum from a young patient administered the same dose. The method was useful for determining the concentration of the enantiomers of amlodipine in hypertension patient serum and for monitoring the transition behavior of the enantiomers in humans. The method proved suitable for application to the separation of the enantiomers of amlodipine and analysis of clinical samples.     

An Automated Multi-Pumping Pulsed Flow System with Spectrophotometric Detection for the Determination of Phosphate in Natural Waters

In this work, an automated multipumping pulsed flow system was implemented for the determination of phosphate in natural waters. The developed flow approach was based on the spectrophotometric determination of phosphate by using the vanadomolybdate reaction. The exploitation of a very simple manifold configuration relying on the utilization of just two active components, in this case two solenoid actuated micropumps that were accountable for sample and reagent insertion and commutation, reaction zone formation, and solutions propelling, provided a great operational and optimization simplicity, low reagent consumption, and waste minimization.

Linear calibration plots for phosphate concentrations of up to 20 mg L^?1 (R² = 0.999, n = 6) were obtained, with a detection limit of 0.2 mg L^?1. The sampling rate was about 60 samples per hour. The system was applied to the monitoring of phosphate in local streams at specific sampling stations.     

Study on the Enantioselective Degradation of Imazethapyr in Soil by CE

A method was developed for the separation of imazethapyr enantiomers using capillary electrophoresis and large volume injection. Optimized buffer conditions were found using 6% hydroxypropyl-β-cyclodextrin as the chiral selector at pH 11 with an injection time of 20 s (0.5 psi). The limits of detection (LOD, S/N = 3) were 0.22 and 0.23 mg L^?1 for the two imazethapyr enantiomers (imazethapyr-I and imazethapyr-II) respectively. The relative standard deviation was less than 5%. This method was successfully used in the study of enantioselective degradation of this herbicide in field soil and the results suggested that imazethapyr-I degraded at a higher rate when compared with imazethapyr-II.