Glycosylated platycosides: Identification by enzymatic hydrolysis and structural determination by LC–MS/MS

In this study, enzymatic hydrolysis and chemometric methods were utilized to discriminate glycosylated platycosides in the extract of Platycodi Radix by LC–MS. Laminarinase, whose enzymatic activity was evaluated using gentiobiose and laminaritriose, was a suitable enzyme to identify the glycosylated platycosides. The laminarinase produced deapi‐platycodin D and platycodin D from the isolated deapi‐platycoside E and platycoside E through the loss of two glucose units by enzymatic reaction, respectively. After hydrolyzing a crude extract by laminarinase, the reconstructed total ion chromatogram generated by a chemometric technique sorted peaks of deglycosylated platycosides easily. Structural information of the glycosylated isomers was revealed through fragment ions generated by the sodiated C_0β ion corresponding to reduced disaccharides in the positive MS⁴ spectra. Characteristic fragment ions of Glc‐(1→6)‐Glc moieties were observed through ring cleavages of ^0,2A_0β, ^0,3A_0β, and ^0,4A_0β, whereas Glc‐(1→3)‐Glc moieties produced only ^0,3A_0β ions. Lithium‐adducted platycosides allowed more detailed structural analysis of glycosidic bond cleavage corresponding to Y_1β and B_1β in addition to ring cleavage.     

Simultaneous determination of multiple platycosides with a single reference standard in Platycodi Radix by high‐performance liquid chromatography coupled with evaporative light scattering detection

A traditional external standard method using HPLC coupled with evaporative light scattering detection has been developed for fast and accurate determination of seven platycosides in Platycodi Radix. However, inevitable difficulties in reference standards preparation process, which are quite costly and time consuming, have made its application limited. To avoid this inconvenience, a simultaneous determination of multiple components with a single reference standard strategy, which could be realized by calibrating the standard curve with internal standard and response factors, was introduced to the HPLC coupled with evaporative light scattering detection method. This is the first time that an incorporation of these two methods has been realized. Among seven ingredients, platycodin D was selected as the internal standard for its relatively easy preparation and low cost. Moreover, according to the investigation on concentration‐dependent effects over response factors and robustness test, platycoside E, deapioplatycodin D, platycodin D, and polygalacin D₂ were chosen to be the indicators for this novel method. The present method has not shown statistically significant differences with a traditional external standard method as verified sample analysis by the F‐test (p = 95%, n = 6).     

New triterpenoid saponins from the roots of Platycodon grandiflorum

Bioassay-directed fractionation of the antiviral active fraction of the roots of Platycodon grandiflorum leads to the isolation of three new triterpenoid saponins, platycosides G1-G3 (1-3), as well as two known saponins, platycodin D3 (4), and platycoside E (5). The structures of the new compounds were elucidated on the basis of their spectral data and chemical evidences. The isolated saponins were tested for their antiviral activities against respiratory syncytial virus (RSV), herpes simplex type 1 virus (HSV-1) and influenza type A virus (Flu A). Compound 4 showed weak anti-RSV activity.     

Separation and identification of enzymatically prepared dephosphorylated products of myo‐inositolhexakisphosphate using LC‐MS

LC‐MS technique described here is a new way for the separation and direct determination of UV–Vis insensitive inositol phosphates (InsP₂‐InsP₆). This circumvents the need of radioisotopic labeling and post‐column derivatization techniques. The method involves separation of various enzymatically dephosphorylated derivatives of InsP₆ on C₁₈‐column using MeOH/H₂O (30:70 v/v) and their identification using electron spray ionization MS in positive ion mode (+pESI‐MS). The LC‐MS studies revealed that the purified phytase from Aspergillus niger van Teighem hydrolyzes InsP₆ in a sequential manner leading to InsP₂ (InsP₂·2Na, t_R 4.4–4.54 min, base peak m/z 382.9) as the end product.     

MALDI Imaging Assisted Discovery of a Di-O-glycosyltransferase from Platycodon grandiflorum Root

A matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) assisted genome mining strategy was developed for the discovery of glycosyltransferase (GT) from the root of Platycodon grandiflorum. A di-O-glycosyltransferase PgGT1 was discovered and characterized that is capable of catalyzing platycoside E (PE) synthesis through the attachment of two β-1,6-linked glucosyl residues sequentially to the glucosyl residue at the C3 position of platycodin D (PD). Although UDP-glucose is the preferred sugar donor for PgGT1, it could also utilize UDP-xylose and UDP-N-acetylglucosamine as weak donors. Residues S273, E274, and H350 played important roles in stabilizing the glucose donor and positioning the glucose in the optimal orientation for the glycosylation reaction. This study clarified two key steps involved in the biosynthetic pathway of PE and could greatly contribute to improving its industrial biotransformation.     

Simultaneous RP-HPLC Determination of Six Platycosides: Application to an Enzymatic Preparation Study

Platycosides, main pharmacological effective compounds, are known to have several biological activities, including anti-obesity, anti-cancer and anti-diabetes. Although enzymatic preparation of platycosides was considered as effective method to obtain them, few analytical methods have been reported on process control. In the present study, we developed an application of reversed-phase high-performance liquid chromatography (RP-HPLC) method for simultaneous determination of six platycosides during the process of enzymatic preparation of deapio-platycodin D (dPD) and platycodin D (PD). The method employed a Hypersil ODS2 analytical column (250 × 4.6 mm I.D., 5 μm) coupled with UV detector at 210 nm with flow rate of 1.0 mg mL⁻¹. A step gradient of acetonitrile–water (v/v) was applied, leading to a sample analysis of 60 min. The method was validated in terms of linearity, sensitivity, precision and accuracy. The correlation coefficients (R ²) for calibration curves of platycosides were in the range of 0.9995–1.0 when the linearity range was from 0.85 to 10.2 mg mL⁻¹. The proposed RP-HPLC method was successfully applied to the analysis of enzymatic preparation study and the recoveries of platycosides were in the range of 96.22–102.56% with RSD <3.3%. The method could be of use for rapid and routine evaluation of the quantity of platycosides during the enzymatic preparation process.

     

Isolation and purification of alkaloids from lotus leaves by ionic‐liquid‐modified high‐speed countercurrent chromatography

An effective high‐speed countercurrent chromatography method was successfully established by using ionic liquids as the modifier of the two‐phase solvent system. Adding a small amount of ionic liquids significantly shortens the separation time and improves the separation efficiency. The conditions of ionic‐liquid‐modified high‐speed countercurrent chromatography including solvent systems, types and content of added ionic liquids, and ionic liquids posttreatment were investigated. The established method was successfully applied to separate alkaloids from lotus leaves using a two‐phase solvent system composed of petroleum ether/ethyl acetate/methanol/water/[C₄mim][BF₄] (1:5:1:5:0.15, v/v/v/v/v). Four alkaloids pronuciferine (1.7 mg), N‐nornuciferine (4.3 mg), nuciferine (3.1 mg), and roemerine (2.1 mg) were obtained with the purities of 90.53, 92.25, 99.86, and 98.63%, respectively, from 100 mg crude extract of lotus leaves. The results indicated that the ionic‐liquid‐modified high‐speed countercurrent chromatography method was suitable for alkaloid separation from lotus leaves and would be a promising method for the separation of alkaloids from other natural products.     

Influence of Isotope Substitution on Lattice and Spin‐Peierls‐Type Transition Features in One‐Dimensional Nickel Bis‐dithiolene Spin Systems

Four new 1D spin‐Peierls‐type compounds, [D₅]1‐(4′‐R‐benzyl)pyridinium bis(maleonitriledithiolato)nickelate ([D₅]R‐Py; R=F, I, CH₃, and NO₂), were synthesized and characterized structurally and magnetically. These 1D compounds are isostructural with the corresponding non‐deuterated compounds, 1‐(4′‐R‐benzyl)pyridinium bis(maleonitriledithiolato)nickelate (R‐Py; R=F, I, CH₃, and NO₂). Compounds [D₅]R‐Py and R‐Py (R=F, I, CH₃, and NO₂) crystallize in the monoclinic space group P2₁/c with uniform stacks of anions and cations in the high‐temperature phase and triclinic space group P$\bar 1$ with dimerized stacks of anions and cations in the low‐temperature phase. Similar to the non‐deuterated R‐Py compounds, a spin‐Peierls‐type transition occurs at a critical temperature for each [D₅]R‐Py compound; the magnetic character of the 1D S=1/2 ferromagnetic chain for [D₅]F‐Py and the 1D S=1/2 Heisenberg antiferromagnetic chain for others appear above the transition temperature. Spin‐gap magnetic behavior was observed for all of these compounds below the transition temperature. In comparison to the corresponding R‐Py compound, the cell volume is almost unchanged for [D₅]F‐Py and shows slight expansion for [D₅]R‐Py (R=I, CH₃, and NO₂) as well as an increase in the spin‐Peierls‐type transition temperature for all of these 1D compounds in the order of F>I≈CH₃≈NO₂. The large isotopic effect of nonmagnetic countercations on the spin‐Peierls‐type transition critical temperature, T_C, can be attributed to the change in ω₀ with isotope substitution.     

Determination of 25‐hydroxyvitamin D3 in human plasma using HPLC with UV detection based on SPE sample preparation

Interest in the metabolism and physiological action of vitamin D is increased exponentially. The most important metabolites of vitamin D are 25‐hydroxyvitamin and 1,25‐dihydroxyvitamin D₃. The aim of the study was to develop a rapid and simple HPLC method for the measurement of 25‐hydroxyvitamin D₃ in human plasma. A method for the measurement of 25‐hydroxyvitamin D₃ using HPLC with UV detection and investigation into the extraction techniques with regard to stability and recovery are described. For the separation, RP column LiChroCart 125‐4, Purospher RP‐18e, 5 μm, was used. The mixture of methanol and deionized water (95:5 v/v) was used as mobile phase. The analytical performance of this method is satisfactory: the intra‐ and inter‐assay coefficients of variation were below 10%. Quantitative recoveries from spiked plasma samples were between 92.0–103.2%. The LOD was 10 nmol/L. The preliminary reference range of 25‐hydroxyvitamin D₃ in a group of blood donors is 62 ± 26 nmol/L.     

Bioactivity‐integrated ultra‐performance liquid chromatography/quadrupole time‐of‐flight mass spectrometry for the identification of nuclear factor‐κB inhibitors and β2 adrenergic receptor agonists in Chinese medicinal preparation Chuanbeipipa dropping pills

A simple and dual‐target method based on ultra‐performance liquid chromatography/quadrupole time‐of‐flight mass spectrometry combined with dual‐bioactive [nuclear factor‐κB (NF‐κB) and β₂‐adrenergic receptor] luciferase reporter assay systems was developed to rapidly characterize the chemical structure of various bioactive compounds of TCM preparations. Chuanbeipipa dropping pills, a traditional Chinese medicine preparation used for the clinical therapy of chronic obstructive lung disease and cough caused by bronchial catarrh, was analyzed with this method. Potential anti‐inflammatory and spasmolytic constituents were screened using NF‐κB and β₂‐adrenergic receptor activity luciferase reporter assay systems and simultaneously identified according to the time‐of‐flight mass spectrometry data. One β₂‐adrenergic receptor agonist (ephedrine) and two structural types of NF‐κB inhibitors (platycosides derivatives and ursolic acid derivatives) were characterized. Platycodin D3 and E were considered new NF‐κB inhibitors. Further cytokine and chemokine detection confirmed the anti‐inflammatory effects of the potential NF‐κB inhibitors. Compared with conventional fingerprints, activity‐integrated fingerprints that contain both chemical and bioactive details offer a more comprehensive understanding of the chemical makeup of plant materials. This strategy clearly demonstrated that multiple bioactivity‐integrated fingerprinting is a powerful tool for the improved screening and identification of potential multi‐target lead compounds in complex herbal medicines. Copyright © 2013 John Wiley & Sons, Ltd.     

Ring‐opening polymerization of benzylated 1,6‐anhydro‐β‐D‐lactose and specific biological activities of sulfated (1→6)‐α‐D‐lactopyranans

A new anhydro disaccharide monomer, 1,6‐anhydro‐2,3‐di‐o‐benzyl‐4‐o‐(2′,3′,4′,6′‐tetra‐o‐benzyl‐β‐D ‐galactopyranosyl)‐β‐D ‐glucopyranose (benzylated 1,6‐anhydro lactose (LSHBE)), was synthesized from D ‐lactose to investigate the polymerizability and biological activities of the resulting branched polysaccharides. The ring‐opening polymerization of LSHBE was carried out with phosphorus pentafluoride as a catalyst under high vacuum to give a stereoregular benzylated (1 → 6)‐α‐D ‐lactopyranan. The molecular weights of poly(LSHBE)s increased with an increase in the amount of CH₂Cl₂ solvent, and polymerization temperatures were affected in both molecular weights and yields of the polymers. The copolymerization of LSHBE with benzylated 1,6‐anhydro‐β‐D ‐glucopyranose (LGTBE) gave the corresponding copolysacchrides having different proportions of lactose and glucose units in good yields. After debenzylation to recover hydroxyl groups and then sulfation, sulfated homopoly(lactose)s and copoly(lactose and glucose)s were obtained. Sulfated homopoly(lactose)s had moderate anti‐HIV (EC₅₀ = 5.9 and 1.3 μg/mL) and blood anticoagulant activities (AA = 18 and 13 unit/mg), respectively. Sulfated copoly(lactose and glucose) having 15 mol % lactose units gave high anti‐HIV and blood anticoagulant activities of 0.3 μg/mL and 54 unit/mg, respectively. These biological results suggest that the distance between branched units on the main chain plays an important role in the anti‐HIV and blood anticoagulant activities. © 2008 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 47: 913–924, 2009     

A new triterpenoid saponin from the roots of Platycodon grandiflorum

A new triterpenoid saponin, 3-O-β-D-glucopyranosyl 16-oxo-platycodigenin 28-O-β-D-apiofuranosyl-(1→3)-β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyra-nosyl ester, was isolated from the roots of Platycodon grandiflorum, together with three known saponins, including platycodin D, deapio platycoside E and platycoside E. The structure of the new compound, named 16-oxo-platycodin D, was elucidated on the basis of spectroscopic data.     

NIR‐Absorbing Donor–Acceptor Based 1,1,4,4‐Tetracyanobuta‐1,3‐Diene (TCBD)‐ and Cyclohexa‐2,5‐Diene‐1,4‐Ylidene‐Expanded TCBD‐Substituted Ferrocenyl Phenothiazines

A series of unsymmetrical (D‐A‐D₁, D₁‐π‐D‐A‐D₁, and D₁‐A₁‐D‐A₂‐D₁; A=acceptor, D=donor) and symmetrical (D₁‐A‐D‐A‐D₁) phenothiazines ( 4 b , 4 c , 4 c′ , 5 b , 5 c , 5 d , 5 d′ , 5 e , 5 e′ , 5 f , and 5 f′ ) were designed and synthesized by a [2+2] cycloaddition–electrocyclic ring‐opening reaction of ferrocenyl‐substituted phenothiazines with tetracyanoethylene (TCNE) and 7,7,8,8‐tetracyanoquinodimethane (TCNQ). The photophysical, electrochemical, and computational studies show a strong charge‐transfer (CT) interaction in the phenothiazine derivatives that can be tuned by varying the number of TCNE/TCNQ acceptors. Phenothiazines 4 b , 4 c , 4 c′ , 5 b , 5 c , 5 d , 5 d′ , 5 e , 5 e′ , 5 f and 5 f′ show redshifted absorption in the λ =400 to 900 nm region, as a result of a low HOMO–LUMO gap, which is supported by TD‐DFT calculations. The electrochemical study exhibits reduction waves at low potential due to strong 1,1,4,4‐tetracyanobuta‐1,3‐diene (TCBD) and cyclohexa‐2,5‐diene‐1,4‐ylidene‐expanded TCBD acceptors. The incorporation of cyclohexa‐2,5‐diene‐1,4‐ylidene‐expanded TCBD stabilized the LUMO energy level to a greater extent than TCBD.     

Efficient Separation of cis‐ and trans‐1,2‐Dichloroethene Isomers by Adaptive Biphen[3]arene Crystals

Reported here is the highly efficient separation of industrially important cis‐ and trans‐1,2‐dichloroethene (cis‐DCE and trans‐DCE) isomers by activated crystalline 2,2′,4,4′‐tetramethoxyl biphen[3]arene (MeBP3) materials, MeBP3α. MeBP3 can be synthesized in excellent yield (99 %), and a cyclic pentamer is also obtained when using 1,2‐dichloroethane as the solvent. The structure of MeBP3 in the CH₃CN@MeBP3 crystal displays a triangle‐shape topology, forming 1D channels through window‐to‐window packing. Desolvated crystalline MeBP3 materials, MeBP3α, preferentially adsorb cis‐DCE vapors over its trans isomer. MeBP3α is able to separate cis‐DCE from a 50:50 (v/v) cis/trans‐isomer mixture, yielding cis‐DCE with a purity of 96.4 % in a single adsorption cycle. Single‐crystal structures and powder X‐ray diffraction patterns indicate that the uptake of cis‐DCE triggers a solid‐state structural transformation of MeBP3, suggesting the adaptivity of MeBP3α materials during the sorption process. Moreover, the separation can be performed over multiple cycles without loss of separation selectivity and capacity.     

Preparative separation of anti‐oxidative constituents from Rubia cordifolia by column‐switching counter‐current chromatography

An effective column‐switching counter‐current chromatography (CCC) protocol combining stepwise elution mode was successfully developed for simultaneous and preparative separation of anti‐oxidative components from ethyl acetate extract of traditional Chinese herbal medicine Rubia cordifolia. The column‐switching CCC system was interfaced by a commercial low‐pressure six‐port switching valve equipped with a sample loop, allowing large volume introduction from the first dimension (1^st‐D) to the second dimension (2^nd‐D). Moreover, to extend the polarity window, three biphasic liquid systems composed of n‐hexane/ethyl acetate/methanol/water (1:2:1:2, 2:3:2:3, 5:6:5:6 v/v) were employed using stepwise elution mode in the 1^st‐D. By valve switching technique the whole interested region of 1^st‐D could be introduced to second dimension for further separation with the solvent system 5:5:4:6 v/v. Using the present column‐switching CCC protocol, 500 mg of crude R. cordifolia extract were separated, producing milligram‐amounts of four anti‐oxidative components over 90% pure. Structures of purified compounds were identified by ¹H and ¹³C NMR.     

Large‐scale separation of antipsychotic alkaloids from Rauwolfia tetraphylla L. by pH‐zone‐refining fast centrifugal partition chromatography

pH‐zone‐refining centrifugal partition chromatography was successively applied in the large‐scale separation of close R_f antipsychotic indole alkaloids directly from CHCl₃ fraction of Rauwolfia tetraphylla leaves. Two experiments with increasing mass from 500 mg to 3 g of crude alkaloid extracts ( 1 C) of R. tetraphylla were carried out in normal‐displacement mode using a two‐phase solvent system composed of methyl tert‐butyl ether/ACN/water (4:1:5, v/v/v) where HCl (12 mM) was added to the lower aqueous stationary phase as a retainer and triethylamine (5 mM) to the organic mobile phase as an eluter. The two centrifugal partition chromatography separations afforded a total of 162.6 mg of 10‐methoxytetrahydroalstonine ( 1 ) and 296.5 mg of isoreserpiline ( 2 ) in 97% and 95.5% purity, respectively, along with a 400.9 mg mixture of α‐yohimbine and reserpiline ( 3 and 4 ). Further, this mixture was resolved over medium pressure LC using TLC grade silica gel H (average particle size 10 μm), which afforded 160.4 mg of α‐yohimbine ( 3) and 150.2 mg of reserpiline ( 4) in >95% purities. The purity of the isolated antipsychotic alkaloids was analyzed by high‐performance LC and their structures were characterized on the basis of their 1D, 2D NMR and electrospray ionization‐mass spectroscopic data.     

Validated high‐performance liquid chromatography method for the determination of the inhibitor of cancer cell invasion (E)‐N‐benzyl‐6‐[2‐(3, 4‐dihydroxy benzylidene)hydrazinyl]‐N‐methylpyridine‐3‐sulfonamide in rat plasma and its application to pharmacokinetic study

In this study, a reliable method for the quantitation of (E )‐N ‐benzyl‐6‐[2‐(3, 4‐dihydroxy benzylidene)hydrazinyl]‐N ‐methylpyridine‐3‐sulfonamide (JW‐55) in rat plasma was developed and validated using high‐performance liquid chromatography. Plasma samples were deproteinized; sildenafil was used as an internal standard. Chromatographic separation was achieved using a reversed‐phase C₁₈ column. The mobile phase, 0.02 m ammonium acetate buffer:acetonitrile (48:52, v /v), was run at a flow rate of 1.0 mL/min at room temperature, and the column eluent was monitored using an ultraviolet detector at 280 nm. The retention times of JW‐55 and sildenafil were ~5.9 and 7.7 min, respectively. The detection limit of JW‐55 in rat plasma was 0.03 μg/mL. Pharmacokinetic parameters of JW‐55 were evaluated after intravenous and oral administration of JW‐55 (10 mg/kg) in rats. After oral administration, the F value was approximately 73.7%.     

Chemo‐Enzymatic Synthesis of 3‐Deoxy‐β‐D‐ribofuranosyl Purines

9‐(3‐Deoxy‐β‐D ‐erythro‐pentofuranosyl)‐2,6‐diaminopurine ( 6 ) was synthesized by an enzymatic transglycosylation of 2,6‐diaminopurine ( 2 ) with 3′‐deoxycytidine ( 1 ) as a donor of 3‐deoxy‐D ‐erythro‐pentofuranose moiety. This transformation comprises i) deamination of 1 to 3′‐deoxyuridine ( 3 ) under the action of whole cell (E. coli BM‐11) cytidine deaminase (CDase), ii) the phosphorolytic cleavage of 3 by uridine phosphorylase (UPase) giving rise to the formation of uracil ( 4 ) and 3‐deoxy‐α‐D ‐erythro‐pentofuranose‐1‐O‐phosphate ( 5 ), and iii) coupling of the latter with 2 catalyzed by whole cell (E. coli BMT‐4D/1A) purine nucleoside phosphorylase (PNPase). Deamination of 6 by adenosine deaminase (ADase) gave 3′‐deoxyguanosine ( 7 ). Treatment of 6 with NaNO₂ afforded 9‐(3‐deoxy‐β‐D ‐erythro‐pentofuranosyl)‐2‐amino‐6‐oxopurine (3′‐deoxyisoguanosine; 8 ). Schiemann reaction of 6 (HF/HBF₄+NaNO₂) gave 9‐(3‐deoxy‐β‐D ‐erythro‐pentofuranosyl)‐2‐fluoroadenine ( 9 ).     

Flotation/ultrasound‐assisted microextraction followed by HPLC for determination of fat‐soluble vitamins in multivitamin pharmaceutical preparations

Dissolved carbon dioxide flotation–emulsification microextraction technique coupled with high‐performance liquid chromatography was developed for separation and determination of fat‐soluble vitamins (A, D₃, E, and K₃) in multivitamin pharmaceutical preparations. Dissolved carbon dioxide flotation was used to break up the emulsion of extraction solvent in water and to collect the extraction solvent on the surface of aqueous sample in narrowed capillary part of extraction cell. Carbon dioxide bubbles were generated in situ through the addition of 300 μL of concentrated hydrochloric acid into the alkaline sample solution at pH = 11.5 (1% w/v sodium carbonate), which was sonicated to intensify the carbon dioxide bubble generation. Several factors affecting the extraction process were optimized. Under the optimal conditions, the limits of detection were 0.11, 0.47, 0.20 and 0.35 μg/L for A, E, D₃, and K₃ vitamins in water samples, respectively. The inter‐day and intra‐day precision of the proposed method were evaluated in terms of the relative standard deviation and were <10.5%.     

Development and validation of a reversed‐phase HPLC method for the determination of γ‐tocotrienol in rat and human plasma

γ‐Tocotrienol (γ‐T3) is a member of the vitamin E family. Recently, γ‐T3 has attracted the attention of the scientific community due to its potent anticancer activity and other therapeutic benefits. The objective of this study was to develop and validate a simple and practical reversed‐phase HPLC method with satisfactory sensitivity for the routine quantification of γ‐T3 in rat and human plasma. The separation of γ‐T3 from the plasma components was achieved with a C₁₈ reversed‐phase column with an isocratic elution using a mixture of methanol, ethanol and acetonitrile (85:7.5:7.5, v/v/v) with a UV detection at 295 nm. γ‐T3 was extracted from rat and human plasma by liquid–liquid extraction with an average recovery of 60%. The method proved linear in the range 100–5000 ng/mL. The inter‐day precision ranged from 5.8 to 12.8% and the accuracy ranged from 92.4 to 108.5%, while the intra‐day precision ranged from 0.7 to 7.9% in both rat and human plasma. This data confirm that the developed method has a satisfactory sensitivity, accuracy and precision for the quantification of γ‐T3 in plasma. To assess its applicability the method was successfully applied to the quantitative analysis for pharmacokinetic studies of γ‐T3 in rats administered a 10 mg/kg single oral dose. Copyright © 2010 John Wiley & Sons, Ltd.