Determination of total thyroxine in human serum by hollow fiber liquid-phase microextraction and liquid chromatography-tandem mass spectrometry.

Determination of total thyroxine in human serum using hollow fiber liquid-phase microextraction (HF-LPME) has been accomplished for the first time. HF-LPME serves as an inexpensive sample pretreatment and the cleanup method that is nearly solvent-free. Thyroxine was extracted through a water immiscible organic solvent immobilized in the wall pores of a polypropylene hollow fiber into 20µl of an aqueous acceptor phase inside the lumen of the hollow fiber. This technique produced extracts that had comparable cleanness with those obtained using solid-phase extraction (SPE). Serum samples with endogenous thyroxine were spiked with isotopically-labeled thyroxine and analyzed by liquid chromatography-tandem mass spectrometry after HF-LPME extraction. Extraction parameters including the organic phase, acid/base concentration of acceptor phase, stirring speed and extraction time were optimized. The calibration range was found to be linear over 1-1000ng/g with the limit of detection (LOD) of 0.3 ng/g. For quantification of total thyroxine in human serum, 6 subsamples were prepared and the results indicated very good precision with a relative standard deviation of <1.3%. The difference from the SPE method was less than 1.2%, with independent t-test showing insignificant bias. Two reference materials of human serum were analyzed, and our obtained values were compared with the reference values. The results showed very good precision with RSD around 0.2% and the deviation from the reference values were -3.1% and -2.1%. The newly developed method is precise, accurate, inexpensive, and environmentally friendly.

Anti-ruthenium antibodies mimic macro-TSH in electrochemiluminescent immunoassay.

BACKGROUND: Macro-hormones are circulating conjugates of hormones with immunoglobulins, which often artefactually elevate biochemical test results. Particularly when causing only moderate elevation no suspicion will be raised. By far the most frequently encountered macro-hormone is macro-prolactin. Here we report a female patient with rheumatoid arthritis who had persistently and grossly elevated thyroid stimulating hormone (TSH) but normal free thyroxine in electrochemiluminescent assays. Although clinically euthyroid, she was put on thyroxine therapy which caused hyperthyroid symptoms. METHODS: An analytic interference by macro-TSH was assumed by dilution experiments, polyethylene-glycol-precipitation, the addition of a heterophilic antibody blocking reagent and size exclusion chromatography. RESULTS: Further workup, however, revealed the presence of anti-ruthenium antibodies. CONCLUSIONS: To our knowledge this is the first report of anti-ruthenium antibodies selectively interfering with a TSH assay and causing erratic gross elevation of TSH mimicking macro-TSH.

Stir bar sorptive extraction combined with high performance liquid chromatography-ultraviolet/inductively coupled plasma mass spectrometry for analysis of thyroxine in urine samples.

tIn this work, polyethyleneglycol (PEG)/hydroxyl polydimethylsiloxane (OH-PDMS)/γ -mercaptopropyltrimethoxysilane (γ -MPTS) coated stir bar was prepared by sol­gel process and its extraction performance for the extraction of amphoteric thyroxines (3,3',5,5'-tetraiodothyronin, T(4); 3,3',5-triiodothyronine, T(3); reversed-3,3',5-triiodothyronine, rT(3)) and their metabolite (3,5-diiodothyronine,T2) was studied. The preparation reproducibility of PEG/OH-PDMS/γ -MPTS coated stir bar was investigated, and the relative standard deviations (RSDs) in the same batch and among different batches were 3.3­14.3% (n = 5) and 7.7­16.6% (n = 3), respectively. The prepared PEG/OH-PDMS/γ -MPTS coated stir bar could be reused for more than 20 times. Based on this fact, a novel method of stir bar sorptive extraction (SBSE) combined with high performance liquid chromatography (HPLC)-ultraviolet (UV)and HPLC-inductively coupled plasma mass spectrometry (ICP-MS) for the analysis of target thyroxinesin human urine samples was developed. The influencing factors of SBSE, such as sample pH, extraction time, stirring rate, salt effect, desorption solution and desorption time, were studied in detail, and the analytical performance of the proposed method was evaluated under the optimized conditions. The enrichment factors (EFs) of the developed method for four target thyroxines were in the range of 14.9­70.4(theoretical enrichment factor was 100). The RSDs were ranging from 4.0% to 13.8% for SBSE-HPLC-UV (c = 25 µg/L, n = 6) and from 3.7% to 6.1% for SBSE-HPLC-ICP-MS (c = 0.5 µg/L, n = 5). The linear range obtained by SBSE-HPLC-UV was 2­500 µg/L for T(2)and 5­500 µg/L for rT3, T(3)and T(4), with correlation coefficients (r) ranging from 0.9957 to 0.9998, respectively, while the linear range obtained by SBSE-HPLC-ICP-MS was 0.05­500 µg/L for T(2) and rT(3), 0.10­200 µg/L for T(3) and 0.05­200 µg/L for T(4)with r ranging from 0.9979 to 0.9998, respectively. The limits of detection (LODs) for the target thyroxines were 0.60­2.20 µg/L for SBSE-HPLC-UV and 0.0071­0.0355 µg/L SBSE-HPLC-ICP-MS, respectively. The developed method was applied for the determination of target thyroxines in urine samples, and the recovery for the spiking samples obtained by SBSE-HPLC-UV was in the range of 81.6­137.6% for human urine,while the recovery for the spiking urine samples obtained by SBSE-HPLC-ICP-MS were in the range of 72.0­121.5%.

Molecular recognition using corona phase complexes made of synthetic polymers adsorbed on carbon nanotubes.

Understanding molecular recognition is of fundamental importance in applications such as therapeutics, chemical catalysis and sensor design. The most common recognition motifs involve biological macromolecules such as antibodies and aptamers. The key to biorecognition consists of a unique three-dimensional structure formed by a folded and constrained bioheteropolymer that creates a binding pocket, or an interface, able to recognize a specific molecule. Here, we show that synthetic heteropolymers, once constrained onto a single-walled carbon nanotube by chemical adsorption, also form a new corona phase that exhibits highly selective recognition for specific molecules. To prove the generality of this phenomenon, we report three examples of heteropolymer-nanotube recognition complexes for riboflavin, L-thyroxine and oestradiol. In each case, the recognition was predicted using a two-dimensional thermodynamic model of surface interactions in which the dissociation constants can be tuned by perturbing the chemical structure of the heteropolymer. Moreover, these complexes can be used as new types of spatiotemporal sensors based on modulation of the carbon nanotube photoemission in the near-infrared, as we show by tracking riboflavin diffusion in murine macrophages.

Direct enantiomer separation of thyroxine in pharmaceuticals using crown ether type chiral stationary phase.

Thyroxine is the tyrosine based hormone produced by the thyroid gland, containing a chiral center in its molecular structure. Synthetic thyroxine is commercially available to treat thyroid dysfunctions, but only a few direct analytical methods to determine its optical purity have been reported. This study attempted to find an optimized liquid chromatographic condition for direct enantiomer separation on crown ether type chiral stationary phase. Among various mobile phases tested, 100% methanol solution containing 10 mM H(2)SO(4) was found to be most appropriate. This chromatographic method was validated and applied to measure the optical purity of six different L-thyroxine products from three domestic pharmaceutical companies and four commercially available D- and L-thyroxine reagents. The chromatographic results showed that every pharmaceutical product had quite high optical purity of above 97% (all but one were higher than 99%) while two of four thyroxine reagents demonstrated relatively higher enantiomer impurity (5-6%).

Enatioselective quantitative separation of D- and L-thyroxine by molecularly imprinted micro-solid phase extraction silver fiber coupled with complementary molecularly imprinted polymer-sensor.

Thyroxine is a known disease biomarker which demands a highly sensitive and selective technique to measure ultratrace level with enantiodifferentiation of its optical isomers (d- and l-), in real samples. In this work, an approach of hyphenation between molecularly imprinted micro-solid phase extraction and a complementary molecularly imprinted polymer-sensor was adopted for enantioseparation, preconcentration, and analysis of d- and l-thyroxine. In both techniques, the same imprinted polymer, coated on a vinyl functionalized self-assembled monolayer modified silver wire, was used as the respective extraction fiber as well as sensor material. This combination enabled enhanced preconcentration of test analyte substantially so as to achieve the stringent limit [limit of detection: 0.0084 ng mL(-1), RSD=0.81%, S/N=3 (d-thyroxine); 0.0087 ng mL(-1), RSD=0.63%, S/N=3 (l-thyroxine)] of clinical detection of thyroid-related diseases, without any problems of non-specific false-positive contribution and cross-reactivity.

Enantiorecognition of triiodothyronine and thyroxine enantiomers using different chiral selectors by HPLC and micro-HPLC.

This paper deals with the chiral separation of triiodothyronine (T3) and thyroxine (T4) by HPLC and micro-HPLC. The separation of T3 and T4 is of great pharmaceutical and clinical interest, since the enantiomers exhibit different pharmacological activities. The HPLC measurements were performed on a chiral stationary ligand-exchange phase using l-4-hydroxyproline bonded via 3-glycidoxypropyltrimethoxysilane to silica gel as a selector. Also a chiral teicoplanin (Chirobiotic) phase was used. In micro-HPLC the chiral separation behaviour of l-4-hydroxyproline, and of the macrocyclic antibiotics teicoplanin and teicoplanin aglycone was investigated for the enantioseparation of T3 and T4. l-4-Hydroxyproline was bonded to 3 microm and the glycopeptide antibiotics were bonded to 3.5 microm silica gel and separations were accomplished by microbore HPLC columns (10 cmx1 mm I.D.). With both techniques and all chiral selectors investigated T3 and T4 were baseline resolved. micro-HPLC was found to be superior to analytical HPLC with respect to low consumption of packing material, mobile phase and analyte.

Enantioselective resolution of thyroxine hormone by high-performance liquid chromatography utilizing a highly fluorescent chiral tagging reagent.

A highly fluorescent chiral tagging reagent, 4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N,N-dimethylaminosulfonyl-2,1,3-benzoxadiazole, [R(-)-DBD-PyNCS], was employed to develop an indirect resolution method for efficient separation of thyroxine enantiomers,D-T(4) and L-T(4). The reaction of R(-)-DBD-PyNCS with the thyroxine enantiomers proceeds effectively at 40 degrees C for 20 min in the presence of basic medium to produce the corresponding pair of diastereomers. No racemization occurs during the tagging reaction under the optimized conditions. Various experimental parameters for derivatization reaction including the species of catalyst, the concentration of tagging reagent and reaction temperatures, have been examined to get a highest yield for T(4) derivatives. The structure of T(4) derivatives was identified based on ESI-MS/MS measurements in negative mode. The efficient separation of D-, L-T(4) derivatives was achieved by isocratic elution with water-acetonitrile mobile phase containing 1% AcOH on a reversed phase column utilizing a conventional fluorescence detector. The resolution (Rs) value of the diastereomers derived from thyroxine was 5.1. The calibration curves of both the D-T(4) and L-T(4) were linear over the concentration range of 0.1-20 microg/ml. The limits of detection (S/N = 3) for both D-T(4) and L-T(4) were 0.2 ng per injection. The proposed method was applied to the determination of D-T(4) and L-T(4) in pharmaceutical formulations and human serum samples.

Direct separation and quantitative analysis of thyroxine and triiodothyronine enantiomers in pharmaceuticals by high-performance liquid chromatography.

A rapid reversed-phase type HPLC method for the simultaneous separation and analysis of D- and L-thyroxine (D- and L-T(4)) and triiodothyronine (T(3)) was developed using a quinine-derived chiral stationary phase and applied for a quantitative assay of the enantiomeric impurity of the drugs in pharmaceutical formulations of levothyroxine. The influence of operating parameters has been studied for the optimization of the separation and also in order to gain an insight into the retention mechanism. Validation of the method included linearity, precision and accuracy which revealed R.S.D. values of <3.3% for intra-assay precision and percent error ranging from -6 to +2.1% for various defined validation samples, proving satisfactory accuracy. Quantitation was performed over the range of 0.5-500 microg ml(-1) with limits of detection and quantitation lower than 0.1 and 0.5 microg ml(-1), respectively, for both analytes. Further, the determination of 0.1% impurity, of D-T(4) as well as L- and D-T(3) in levothyroxine sodium tablets proved to be feasible.

Effect of acidity on the enantiomeric resolution of thyroxine and tocainide by HPLC on a (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid column.

Enantiomeric resolution of thyroxine and tocainide was achieved on a (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid column. The mobile phases were methanol/water (4:1, v/v) and methanol/water containing 5 mM sulfuric acid (4:1, v/v) for tocainide and thyroxine respectively. The flow rate was 0.5 ml/min. The effect of the acidity on the chiral resolution of these drugs was studied. Detection was at 220 nm for both drugs. The values of alpha and Rs were 2.08-3.11 and 1.00-2.60, respectively, for thyroxine while the values of alpha and Rs were 1.13-1.26 and 0.10-1.30, respectively, for tocainide.

[Determination of thyroxine enantiomers in human plasma with normal high performance liquid chromatography-chiral complex exchange mobile phase].

An HPLC method was developed with normal HPLC-chiral complex mobile phase for the separation and determination of D- and L-thyroxine enantiomers (D- and L-T4) in human plasma. The method includes extraction of thyroxine from plasma and separation of thyroxine enantiomers on HPLC silica column with chiral eluent containing L-proline, cupric acetate and triethylamine (TEA). The sensitivity of the method was 0.1 mg/L. The precisions of inter-day and intra-day, linearity, extraction recovery, and stability of T4 enantiomers in plasma and in deproteinized plasma were determined for the validation of the method. Baseline enantioseparation of the compounds containing D- and L-T4 was achieved. Meantime we determined the concentrations of D-T4 and L-T4 in plasma of 15 volunteers with euthyroid, hypothyroid and hyperthyroid symptoms. Liquid chromatographic method based on the described procedure was useful for the determination of D- and L-thyroxine in patient plasma and for pharmacokinetics investigation.

Ion-pair high-performance liquid chromatographic separation of two thyroxine glucuronides formed by rat liver microsomes.

A simple reversed-phase ion-pair high-performance liquid chromatographic separation method has been developed for thyroxine (T4) and its glucuronide metabolites formed by liver microsomes of untreated and 3-methylcholanthrene-treated rats. Besides the phenol-T4-glucuronide, another, probably acyl-T4-glucuronide, formation has been detected. The effect of pH and temperature on the stability of the acyl-T4-glucuronide was also investigated. The lowering of pH to 2 and cooling the samples to 5 degrees C is necessary to prevent the hydrolysis of acylglucuronide, while both pH and temperature do not affect the stability of the phenol-T4-glucuronide. The retention times of T4 and phenol-T4-glucuronide are highly influenced by the pH of the mobile phase, but not that of acyl-T4-glucuronide.

Optical purity determination of thyroxine enantiomers in bulk materials by chiral thin layer chromatography.

L-thyroxine and D-thyroxine were separated on ligand exchange chiral thin layer chromatographic plates, using a solvent system consisting of acetonitrile:methanol:water 60:15:15 v/v, at a wavelength of 254 nm. The methodology, chiral recognition mechanism(s) involved and its application are discussed.

Identification of thyroxine-sulfate (T4S) in human serum and amniotic fluid by a novel T4S radioimmunoassay.

Recently, we identified significant amounts of thyroxine sulfate (T4S) in fetal sheep serum, meconium, bile, and amniotic and allantoic fluids. Little is known, however, about sulfate conjugation of thyroxine in humans. In this study, we employed a novel, sensitive T4S RIA to address this question. The rabbit antiserum was quite specific; T4, T3, rT3, and 3,3'-T2 showed less than 0.002% cross-reactivity. Other analogs cross-reacted less than 0.0001%. Only rT3S and T3S cross-reacted significantly (9.9% and 2.0%, respectively). The mean serum T4S concentration (ng/dL) was 8.6 in euthyroid subjects, 14.4 in hyperthyroid subjects, 5.0 in hypothyroid subjects, 5.9 in pregnancy, and 4.5 in patients with nonthyroid illnesses. T4S concentration in amniotic fluid from women at 18-19 weeks of gestation (25.5 ng/dL) was higher than that at 14-15 weeks of gestation (14.3 ng/dL). A significant rise in serum T4S was detected in hyperthyroid patients 1 day after ingestion of 1 g of ipodate. These data suggest that T4S is a normal component of human serum and amniotic fluid, and it is mostly derived from T4 peripherally and accumulates when type I 5'-monodeiodinating activity is low in fetuses or inhibited by drugs, such as ipodate.

Comparison of different methods for iodothyronines extraction from animal tissues.

Four different methods for thyroid hormones concentration in various tissues have been compared. The methods involve homogenization, extraction with 95% ethanol and assay of iodothyronines by a highly sensitive RIA. The method described by Chopra et al. has been selected and used for further estimations as the most reliable and convenient for a continuous laboratory work. The T4 levels (means +/- SE) found in the liver, kidney and skeletal muscle were 22.49 +/- 2.19, 14.89 +/- 1.56 and 8.33 +/- 1.73 ng per g wet tissue. The T3 contents were 8.53 +/- 1.08, 9.52 +/- 0.69 and 4.90 +/- 0.76 ng per g wet tissue, respectively.

Changes in tissue and blood concentrations of thyroid hormones in developing chum salmon.

The changes in tissue and blood concentrations of thyroxine (T4) and triiodothyronine (T3) were examined during development of the chum salmon (Oncorhynchus keta). Extraction methods previously established for tissue T4 were also validated for tissue T3, by parallel displacement curves to T3 standard in the radioimmunoassay and by the same elution patterns of immunoreactivity in a HPLC system. The T3 concentration of the eggs just after fertilization (4-9 ng/g) was lower than the T4 concentration (5-15 ng/g). Both T4 and T3 concentrations in the whole body decreased steadily during yolk absorption, primarily due to the decline of the hormone content in the yolk. Both T4 and T3 were detected in blood plasma at later stages of yolk absorption, and the plasma levels increased toward the end of yolk absorption. At the end of yolk absorption, when the larvae emerge from the gravel bed, a transient increase in whole body concentrations of T4 and T3 was observed. Plasma levels of T4 were always greater than the T3 levels. Thyroid follicles began to develop during the early stages of yolk absorption. These findings suggest important roles of maternal thyroid hormones for developing salmon embryos during yolk absorption.

Improvement in HPLC fractionation of thyroxine-containing thyroglobulin tryptic peptides by prior Accell ion-exchange column chromatography.

The isolation and purification of the peptides containing the hormonogenic tyrosyl residues in thyroglobulin is of great interest to the understanding of structure-function relationships in this iodoprotein. This is usually performed in reduced alkylated selectively hydrolyzed thyroglobulin by subsequent HPLC fractionation. However, the main difficulty encountered when trying to isolate these peptides is their disproportion with respect to the total number of possible peptides (14 vs a total of 508). Several HPLC runs with different mobile phases are necessary and each run is accompanied with significant losses of the peptides to be purified. In an attempt to improve the separation of the T4-containing peptides in thyroglobulin tryptic digests from the much more abundant iodotyrosine-containing ones, which are present as contaminants, we have used a very simple and fast step prior to the HPLC fractionation as it is a self-packed ion-exchange column chromatography. This preliminary step results in an improvement in the separation of these peptides and leads to a relative enrichment of the hormonogenic peptides falling in different zones of the HPLC chromatogram, which facilitates their subsequent separation and purification by HPLC.