Prapin Wilairat

Simultaneous determination of paracetamol and chlorpheniramine maleate by micellar electrokinetic chromatography.

A micellar electrokinetic chromatography (MEKC) method was established for determination of paracetamol (PARA) and chlorpheniramine maleate (CPM) in cold tablets. Separation of both drugs, as well as other seven cold remedy ingredients, was achieved in 25.5 min using a sodium dihydrogenphosphate-sodium tetraborate buffer (10 mM, pH 9.0) containing sodium dodecyl sulfate (SDS) (50 mM) and acetonitrile (26% v/v). The effective capillary length of 50 cm, the separating voltage of 15 kV and the temperature of 30 degrees C was optimized. Detection was by a diode array detector at 214 nm. Method linearity was excellent (r(2)>0.999) over the concentration tested (10-250 microg/ml) with good precision and accuracy. Recoveries were good (>99%) with limits of detection of 0.4 and 0.5 microg/ml and limits of quantitation of 2 (%R.S.D.=3.1%) and 4 (%R.S.D.=2.4%) microg/ml, for PARA and CPM, respectively. The developed method was applied to the determination of ingredients in cold tablets and was found to be simple, rapid and efficient.

Pervaporation-flow injection with chemiluminescence detection for determination of iodide in multivitamin tablets

This paper describes the use of a pervaporation (PV) technique in a flow injection (FI) system for selective improvement in iodide analysis. Iodide in the sample zone is oxidized to iodine, which permeates through a hydrophobic membrane. Detection of the diffused iodine is achieved using the chemiluminescent (CL) emission at 425nm that results from the reaction between iodine and luminol. The method was applied for the analysis of some pharmaceutical products, such as nuclear emergency tablets and multivitamin tablets. Ascorbic acid present in multivitamin samples interfered seriously with the analysis, and off-line sample treatment using anion exchange resin was employed to successfully remove ascorbic acid before the analysis. Ascorbic acid was flushed from the column using 0.4M sodium nitrate followed by elution of iodide with 2M sodium nitrate. The detection limit (3S.D.) of the system was 0.5mgl(-1), with reproducibility of 5.2% R.S.D. at 5mgl(-1). Sample throughput was determined as 30injectionsh(-1). There was good agreement between iodide concentrations from extracted samples determined using four different methods, i.e., PV-FI, gas diffusion-flow injection, potentiometry and ICP-MS. A comparison of the analytical features of the developed pervaporation system with these of the previously reported chemiluminescence gas diffusion-flow injection previously reported is also described.

UGT1A6 genotype-related pharmacokinetics of deferiprone (L1) in healthy volunteers.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT UGT1A6 has been proposed as the predominant isoform responsible for the glucuronidation of deferiprone. UGT1A6*2 allele has been associated with the altered enzyme activity. WHAT THIS STUDY ADDS There is no statistically significant effect of UGT1A6 genotype on the single-dose pharmacokinetics of deferiprone in healthy volunteers. Gender influences serum pharmacokinetics of deferiprone. Body iron stores reflected by serum ferritin levels may have an influence on the extent of extravascular deferiprone distribution. AIMS To examine the effects of UGT1A6 polymorphisms on the pharmacokinetics of deferiprone in healthy volunteers. METHODS Twenty-two healthy volunteers were enrolled and grouped according to UGT1A6 genotype. After an overnight fast, the subjects received a single oral dose of 25 mg kg(-1) deferiprone. Blood samples were collected at 0, 15, 30, 45, 60, 90, 120, 180, 240, 300 and 360 min after dosing. Urine output was collected at 0, 0-2, 2-4, 4-8, 8-12 and 12-24 h. Deferiprone (L1) and deferiprone-glucuronide (L1G) concentrations in serum and urine were determined using a validated high-performance liquid chromatography method. UGT1A6 genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism analysis. RESULTS No statistically significant differences in any pharmacokinetic parameters of either deferiprone or deferiprone-glucuronide among the genotype groups were noted. Likewise, there were no statistically significant differences in 24-h urinary deferiprone and deferiprone-glucuronide excretion among the genotype groups. Significant differences between men and women were found in AUC(0-infinity), V(d)/F, and CL/F of deferiprone. Gender differences in 24-h urinary deferiprone and its metabolite excretion, however, failed to reach statistical significance. The V(d)/F of deferiprone was found to correlate significantly with serum ferritin (r(s) = 0.665; P = 0.001). CONCLUSION The studied single nucleotide polymorphisms in UGT1A6 do not appear to exert statistically significant effects on the single-dose pharmacokinetics of deferiprone. Gender appears to influence the serum pharmacokinetics of deferiprone, but not urinary excretion of deferiprone and its metabolite. Body iron stores may have an influence on the extent of extravascular deferiprone distribution.

Enantiomeric separation of some common controlled stimulants by capillary electrophoresis with contactless conductivity detection

CE methods with capacitively coupled contactless conductivity detection (C4D) were developed for the enantiomeric separation of the following stimulants: amphetamine (AP), methamphetamine (MA), ephedrine (EP), pseudoephedrine (PE), norephedrine (NE) and norpseudoephedrine (NPE). Acetic acid (pH 2.5 and 2.8) was found to be the optimal background electrolyte for the CE‐C4D system. The chiral selectors, carboxymethyl‐β‐cyclodextrin (CMBCD), heptakis(2,6‐di‐O‐methyl)‐β‐cyclodextrin (DMBCD) and chiral crown ether (+)‐(18‐crown‐6)‐2,3,11,12‐tetracarboxylic acid (18C6H4), were investigated for their enantioseparation properties in the BGE. The use of either a single or a combination of two chiral selectors was chosen to obtain optimal condition of enantiomeric selectivity. Enantiomeric separation of AP and MA was achieved using the single chiral selector CMBCD and (hydroxypropyl)methyl cellulose (HPMC) as the modifier. A combination of the two chiral selectors, CMBCD and DMBCD and HPMC as the modifier, was required for enantiomeric separation of EP and PE. In addition, a combination of DMBCD and 18C6H4 was successfully applied for the enantiomeric separation of NE and NPE. The detection limits of the enantiomers were found to be in the range of 2.3–5.7 μmol/L. Good precisions of migration time and peak area were obtained. The developed CE‐C4D method was successfully applied to urine samples of athletes for the identification of enantiomers of the detected stimulants.

Evaluation of World Anti-Doping Agency criteria for anabolic agent analysis by using comprehensive two-dimensional gas chromatography–mass spectrometry

AbstractThis work presents the validation study of the comprehensive two-dimensional gas chromatography (GC×GC)–time-of-flight mass spectrometry method performance in the analysis of the key World Anti-Doping Agency (WADA) anabolic agents in doping control. The relative abundance ratio, retention time, identification and other method performance criteria have been tested in the GC×GC format to confirm that they comply with those set by WADA. Furthermore, tens of other components were identified with an average similarity of >920 (on the 0–999 scale), including 10 other endogenous sterols, and full mass spectra of 5,000+ compounds were retained. The testosterone/epitestosterone ratio was obtained from the same run. A new dimension in doping analysis has been implemented by addressing separation improvement. Instead of increasing the method sensitivity, which is accompanied by making the detector increasingly “blind” to the matrix (as represented by selected ion monitoring mode, high-resolution mass spectrometry (MS) and tandem MS), the method capabilities have been improved by adding a new “separation” dimension while retaining full mass spectral scan information. Apart from the requirement for the mass spectral domain that a minimum of three diagnostic ions with relative abundance of 5% or higher in the MS spectra, all other WADA criteria are satisfied by GC×GC operation. The minimum of three diagnostic ions arises from the need to add some degree of specificity to the acquired mass spectrometry data; however, under the proposed full MS scan method, the high MS similarity to the reference compounds offers more than the required three diagnostic ions for an unambiguous identification. This should be viewed as an extension of the present criteria to a full-scan MS method. FigureImproved separation of AAS in doping control: GC×GC format

Flow-injection determination of iodide ion in nuclear emergency tablets, using boron-doped diamond thin film electrode.

The electrochemical determination of iodide was studied at boron-doped diamond thin film electrodes (BDD) using cyclic voltammetry (CV) and flow-injection (FI) analysis, with amperometric detection. Cyclic voltammetry of iodide was conducted in a phosphate buffer pH 5. Experiments were performed using glassy carbon (GC) electrode as a comparison. Well-defined oxidation waves of the quasi-reversible cyclic voltammograms were observed at both electrodes. Voltammetric signal-to-background ratios (S/B) were comparable. However, the GC electrode gives much greater in the background current as usual. The potential sweep rate dependence exhibited that the peak current of iodide oxidation at 1mM varied linearly (r(2) = 0.998) with the square root of the scan rate, from 0.01 to 0.30Vs(-1). This result indicates that the reaction is a diffusion-controlled process with negligible adsorption on BDD surface, at this iodide concentration. Results of the flow-injection analysis show a highly reproducible amperometric response. The linear working range was observed up to 200muM (r(2) = 0.999). The detection limit, as low as 0.01muM (3sigma of blank), was obtained. This method was successfully applied for quantification of iodide contents in nuclear emergency tablets.

Quantitative determination of ortho- and meta-tyrosine as biomarkers of protein oxidative damage in β-thalassemia

Abstract Oxidative stress in thalassemia is caused by secondary iron overload and stems from blood transfusion and increased iron uptake. In this study, we hypothesized that levels of o- and m-tyrosine, products of hydroxyl radical attack on phenylalanine, would be elevated in β-thalassemia (intermediate). This study represents the first report in which specific markers of protein oxidative damage have been quantified in thalassemia. We used GC/MS to assay o- and m-tyrosine at the femtomole level using only a few microliters of plasma. Levels of both markers were significantly higher in patients with β-thalassemia than in controls and were positively correlated with serum ferritin, malondialdehyde, superoxide dismutase, glutathione peroxidase and glutathione. We conclude that o- and m-tyrosine are useful biomarkers of oxidative damage to proteins in thalassemia (intermediate) and may also be useful markers in other iron overload diseases. Positive correlations between o- and m-tyrosine levels and malondialdehyde as well as antioxidants such as superoxide dismutase, glutathione peroxidase and glutathione, are indicative of the broad impact of oxidative stress on blood plasma in thalassemia, with up-regulation of antioxidant proteins probably reflecting a homeostatic response to these increased stress levels.

HIGH-PERFORMANCE LIQUID CHROMATOGRAPHIC SEPARATION AND QUANTITATION OF STEVIOSIDE AND ITS METABOLITES

A method of analysis using high performance liquid chromatography (HPLC) was developed for the separation and quantitation of the metabolites of stevioside: steviol-16,17α-epoxide, 15α-hydroxysteviol, steviolbioside, isosteviol, and steviol. The separation was carried out on a reversed-phase C18 Nova-Pack column with gradient elution of acetonitrile/water mixture. The applicability of the method was demonstrated in the detection and separation of stevioside and its metabolites found in blood, feces, and urine of hamsters after ingestion of stevioside.

Separation of α‐, β‐, γ‐, δ‐tocopherols and α‐tocopherol acetate on a pentaerythritol diacrylate monostearate‐ethylene dimethacrylate monolith by capillary electrochromatography

This work reports the first use of a monolith with method development for the separation of tocopherol (TOH) compounds by CEC with UV detection. A pentaerythritol diacrylate monostearate‐ethylene dimethacrylate (PEDAS‐EDMA) monolithic column has been investigated for an optimised condition to separate α‐, β‐, γ‐ and δ‐TOHs, and α‐tocopherol acetate (TAc). The PEDAS‐EDMA monolith showed a remarkably good selectivity for separation of the TOH isomers including the β‐ and γ‐isomers which are not easily separated by standard C8 or C18 particle‐packed columns. Retention studies indicated that an RP mechanism was involved in the separation on the PEDAS‐EDMA column, but polar interactions with the underlying ester and hydroxyl groups enhanced the separation of the problematic β‐ and γ‐isomers. Separation of all the compounds was achieved within 25 min using 3:10:87 v/v/v 100 mM Tris buffer (pH 9.3)/methanol/ACN as the mobile phase. The method was successfully applied to a pharmaceutical sample with recoveries from 93 to 99%. Intraday and interday precisions (%RSD) for peak area and retention time were less than 2.3. LODs for all four TOHs and TAc were below 1 ppm.

Pharmacokinetics of deferiprone in patients with β-thalassaemia: impact of splenectomy and iron status.

Background and ObjectiveIron-rich transfusions and/or a compensatory increase in iron absorption ultimately result in iron loading in patients with β-thalassaemia. Hence, without iron chelation, iron accumulates relentlessly. Deferiprone has been shown to be capable of reducing the iron burden in patients with b-thalassaemia. However, there is wide interpatient variation in deferiprone-induced urinary iron excretion (UIE). We hypothesized that splenectomy and iron status might influence the pharmacokinetic profiles of deferiprone in patients with β-thalassaemia/haemoglobin E, and the present study was aimed at examining this hypothesis.Study Participants and MethodsThirty-one patients with β-thalassaemia/haemoglobin E (20 splenecto-mized and 11 non-splenectomized patients) were enrolled in the study. After an overnight fast, the subjects received a single oral dose of deferiprone 25 mg/kg of body weight. Blood samples were collected pre-dosing and at 15, 30, 45, 60, 90, 120, 180, 240, 300, 360 and 480 minutes after dosing. Urine output was pooled and collected at 0–2, 2–4, 4–8, 8–12 and 12–24 hour intervals. Serum and urine concentrations of deferiprone and its metabolite deferiprone glucuronide were determined using a validated high-performance liquid chromatography method. Serum deferiprone-chelated iron and UIE were determined using a validated colourimetric method.ResultsNo significant difference in the pharmacokinetic parameters of non-conjugated deferiprone was observed between splenectomized and non-splenectomized patients. However, the maximum serum concentration (Cmax) and the area under the serum concentration-time curve (AUC) from time zero to infinity (AUC∞) values of deferiprone glucuronide were significantly lower (both p < 0.05) in splenectomized patients (median 53.2µmol/L and 12 634 µmol · min/L, respectively) than in non-splenectomized patients (median 70.5 µmol/L and 20 601 mmol · min/L, respectively). The Cmax and the AUC from time zero to the time of the last measurable concentration (AUClast) values of serum deferiprone-chelated iron, as well as UIE, were significantly higher (p < 0.001) in splenectomized patients (median values 7.1 µmol/L, 1645 mmol · min/L and 77.1 mmol, respectively) than in non-splenectomized patients (median values 3.1 µmol/L, 545 mmol · min/L and 12.5 µmol, respectively). Urinary excretion of non-conjugated deferiprone and deferiprone glucuronide did not differ between the two groups. Further analyses using multiple linear regressions indicated that the iron profiles (non-transferrin-bound iron and ferritin) were significant predictors of the pharmacokinetic parameters of non-conjugated deferiprone, deferiprone-chelated iron and UIE. In addition, splenectomy status was identified as the strongest predictor of the AUClast of deferiprone-chelated iron and UIE.ConclusionBoth iron and splenectomy status have significant effects on the pharmacokinetics and iron chelation efficacy of deferiprone. A greater degree of iron overload in splenectomized patients results in alterations in pharmacokinetic parameters (the Cmax and AUC) of deferiprone glucuronide and deferiprone-chelated iron, as well as a significant increase in UIE.