Separation thyroxine

Purification The crude precipitate is dissolved in boiling 2N HCl (300 ml) and filtered from the relatively insoluble thyroxine hydrochloride. The hot filtrate is brought to pH 4 with 5N NaOH and triiodo thyronine again separates after chilling at 0° to 4°C it is collected, washed with water and dried. The yield of triiodo thyronine is 70 to 75% of the theoretical. This triiodo thyronine still contains some thyroxine (about 10%). 

The final purification consists of chromatographic separation of thyroxine and triiodo thyronine on a kieselguhr column using 20% chloroform in n-butanol equilibrated with... 

Naidook S. Separation of Acetic and Propionic Acid Analogs of I Thyroxine and l ltiiodotKynHiine by Thin-Layer Ctoomatography , AnaL Biochem. 1978,91, 543-547. 

In addition to neutral loss scans, mass spectrometers can be used to detect other compounds in a different manner. Acylcamitines are fatty acid esters of carnitine. The masses of acylcamitines differ by the size of the fatty acid attached to it. The tandem mass spectrometer can detect these selectively as well because they all produce a similar product, in this case an ion rather than a molecule. Because it is an ion, it can be detected by the second mass separation device. The ion has a mass of 85 Da and is common to all acylcamitines. Performing a precursor ion scan of 85 Da (essentially a scan of only molecules that produce the 85 ion) reveals a selective analysis of acylcar-nitines, as shown in Fig. 14.2. Additional scans have been added to more selectively detect basic amino acids, free carnitine, short chain acylcamitines and a hormone, thyroxin (T4) which has amino acid components. 

An optical immunosensor for continuous T4 measurement has been described, in which the fluorescent indicator protein is separated from the sample flow chamber by a dialysis membrane.024) The indicator is T4-binding globulin (TBG), the intrinsic fluorescence (ex. 290 nm) of which is quenched by T4binding. Due to the high affinity of the TBG for thyroxine, the immunosensor is not reversible, but multiple measurements can be made until the TBG is saturated. Sensitivity is inadequate for clinically useful concentrations of T4, but suggestions for improvement of the method are made. 

Two hours is usually sufficient time for the completion of the reaction and the separation of these salts. The products are readily hydrolysed by warm water. The yields from aliphatic and aromatic (other than o-substituted) nitriles are usually quantitative. The method has found an important application in the synthesis of thyroxine (Ann. Rep., 1926, 236.) The details of the following preparation constitute a general method of procedure. 

Iron binds thyroxine in vitro (80) and ferrous sulfate can reduce thyroxine absorption (81). This can result in hypothyroidism (80,81). A similar interaction with ferrous fumarate has been described in a patient who took both medications simultaneously (83). Separation of thyroxine from iron may be prudent, since this interaction may reflect the formation of a non-absorbable complex in the gut (80). 

The final purification consists of chromatographic separation of thyroxine and triiodo thyronine on a kieselguhr column using 20% chloroform in n-butanol equilibrated with 0.5N NaOH as the developing solvent. 80 to 100 mg triiodo thyronine is purified during each run on a 50 g kieselguhr column. Pure L-triiodo thyronine has MP 236° to 237°C (dec.) and [a]D29-5° = +21.5 in a 4.75% solution in a mixture of 1 part of N HCI and 2 parts of ethanol. Liothyronine is commonly used as the sodium salt. 

E4. Endo, Y., Miyai, K., Hata, N. and Ichihara,K., Fractionation of polyclonal antibody by isoeletric focusing and chromatofocusing. Separation of high-affinity rabbit clonotype anti-thyroxine antibody. Anal. Biochem. 144, 41-46 (1985). 

Y4. Yamamoto, R., Hattori, S., Inukai, T., Matsuura, A., Yamashita, K., Kosaka, A., and Kato, K., Enzyme immunoassay for thyroxine and triiodothyronine in human serum, with use of a covalent chromatographic separation method. Clin. Chem. 27, 1721-1723 (1981). 

Thyroxine and thyroxine glucuronide were separated on a C]8 Ultrabase column (4.6 mm X 250 mm, 10 p.m). Solvent A was a 35 65 (v/v) mixture of methanol and 20 mM potassium phosphate buffer (pH 7.0) containing 1% triethylamine. Solvent B was methanol. A linear gradient from 0 to 100% B was run in 15 minutes. Elution was completed by maintaining 100% B for 5 minutes. Two methods of on-line radiochemical detection was used liquid scintillation using a 2 mL detection cell, and solid scintillation using lithium scintillator glass in an effective cell volume of 400 p-L. The latter method was most convenient. 

Interactions. Iron chelates in the gut with tetracyclines, penicillamine, methyldopa, levodopa, carbidopa, ciprofloxacin, norfloxacin and ofloxacin it also forms stable complexes with thyroxine, captopril and biphosphonates. These interactions can be clinically important. Ingestion should be separated by 3 hours. 

It has been applied to the separation and enrichment of organic compounds such as indole auxins, bromoacetyl thyroxine and its analogs, dinitrophenyl amino acids. 

Sample material Serum or plasma (EDTA or heparin). Oxalate and citrate are not suitable as anticoagulants for plasma separation, since the thyroxine concentration may be reduced. Serum and plasma samples can be stored in the refrigerator at +2°C to +8°C for one week. 

Both enantiomers of hormones thyroxine (d- and l-T4, 38) and triiodothyronine (d- and l-T3, 39) can also be baseline separated and quantitatively determined in pharmaceutical formulations of Zevo-thyroxine with the use of commercial t-BuCQN... 

Gika, H., Lammerhofer, M., Papadoyan-nis, I., Lindner,W. Direct separation and quantitative analysis of thyroxine and triiodothyronine enantiomers in pharmaceuticals by high-performance liquid chromatography, J. Chromatogr. B, 2004, 800, 193-201. 

Using bis (trimethylsilyl) acetamide (BSA), volatile trimethylsilyl (TMS) derivatives of the active components 3,5,3, 5 -tetraiodothyronine (thyroxin, TJ and 3,5,3 -triiodothyronine (T3) have been prepared as well as TMS derivatives of the nonphysiologically active components 3,3, 5 -triiodothyronine (T3 ), 3,5-diiodothyronine (T2), and 3,5-diiodo-tyrosine (DIT). Separation and quantitative estimation of these iodinated amino acids is achieved by gas-liquid chromatography. The method is... 

Most of the assays now carried out with isoluminol derivatives are solid-phase competitive assays (Fig. 2), in which the analyte to be determined competes with the labeled analyte for the available binding sites on antibodies that are immobilized on the solid phase. Thus, plastic microspheres have been used to immobilize antibodies for estriol (K12) or thyroxine (W9). Estradiol antibodies have been immobilized onto plastic tubes (K7) or beads (K16). In all of the above cases, the amount of immunoconjugated hormone label is inversely proportional to the amount of free hormone in the analytical sample. Unconjugated labels are removed by decantation or aspiration from the solid phase, and the specific, immunoconjugated labels are measured in a luminometer. This approach has been successfully applied to progesterone analyses in either serum (D6) or saliva (D5). A review of several separation-based assays with isoluminol analogs was presented a few years ago by Kohen et al. (K18). 

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