Identification and quantification of two antihepatotoxic coumarinolignoids cleomiscosin A and cleomiscosin B in the seeds of Cleome viscosa using liquid chromatography-tandem mass spectrometry

A sensitive liquid chromatography/electrospray ionization tandem mass spectrometric (LC/ESI-MS/MS) method was developed for the identification and quantification of two antihepatotoxic coumarinolignoids cleomiscosin A and cleomiscosin B in different extracts of the seeds of Cleome viscosa. The separation of cleomiscosin A and cleomiscosin B was achieved on an RP(18) column using a solvent system consisting of a mixture of acetonitrile-methanol (1:2, v/v) and acetonitrile-water-formic acid (5:95:0.3, v/v) as a mobile phase in a gradient elution mode. A multiple-reaction monitoring (MRM) method was developed for quantification of cleomiscosin A and cleomiscosin B in the seed extracts of Cleome viscosa. On the basis of signal-to-noise ratio of 3, the limit of detection in MRM mode for cleomiscosin A and cleomiscosin B were 1.0 and 4.0 ng/mL respectively. The method was validated in terms of linearity, accuracy and precision for 6 days. The method developed was found to be useful for identification and quantification of cleomiscosin A and cleomiscosin B in the different extracts of the seeds of Cleome viscosa.     

High-performance liquid chromatographic method for identification and quantification of two isomeric coumarinolignoids-cleomiscosin A and cleomiscosin B-in extracts of Cleome viscosa

A simple, accurate and reproducible reverse-phase HPLC method has been developed for identification and quantification of two isomeric coumarinolignoids, cleomiscosin A and B in different extracts of the seeds of Cleome viscosa using photodiode array detection at 326 nm. Cleomiscosin A and B were separated on a Waters symmetry C(18) column (250 x 4.6 mm with 5.0 microm particle size) with an isocratic elution system composed of acetonitrile-methanol (1:2, v/v) and acetic acid-water (0.5:99.5, v/v) in the ratio of 40:60 (v/v). The calibration curves were linear (r(2) > 0.997) in the concentration ranges of 20-100 microg/mL for both compounds. The limits of detection and quantification were 15 and 20 microg/mL for both cleomiscosin A and B. The intra- and inter-day precisions were 3.68 and 2.22% for cleomiscosin A and 4.22 and 5.06% for cleomiscosin B. The recoveries measured at two different concentration levels varied from 98.03 to 110.06%. The method was used to identify and quantify cleomiscosins A and B in different extracts of Cleome viscosa seeds.     

High-performance liquid chromatography and LC-ESI-MS method for the identification and quantification of two biologically active isomeric coumarinolignoids cleomiscosin A and cleomiscosin B in different extracts of Cleome viscosa

A rapid, sensitive and simple reverse-phase high-performance liquid chromatographic-electrospray ionization-mass spectrometry method for simultaneous determination of cleomiscosin A and cleomiscosin B has been developed and validated. The isomeric coumarinolignoids cleomiscosin A (1) and cleomiscosin B (2) were separated on a Waters symmetry C(18) column with a solvent system composed of acetonitrile-methanol (1:2) and acetic acid-water (0.5:99.5) in a gradient elution mode. The absorption at 326 nm was chosen as the measuring wavelength in which resolution and baseline separation of compounds 1 and 2 could be obtained. The identity of the two isomeric compounds 1 and 2 in the samples were determined on a triple quadrupole mass spectrometer with ESI interface operating in the positive mode. Calibration curves were linear (r(2) > 0.993) over the concentration range 20-200 microg/mL for cleomiscosin A and 10-200 microg/mL for cleomiscosin B with acceptable accuracy and precision, respectively. The intra-day and inter-day precision were 1.13 and 0.82% for cleomiscosin A and 1.78 and 1.28% for cleomiscosin B, respectively. The validated method was successfully applied for the analysis of the above two compounds in different extracts of Cleome viscosa.     

Chromatographic method for clobetasol propionate determination in hair follicles and in different skin layers

Clobetasol propionate (CLO) is a potent steroid used for the treatment of several dermatological diseases. Recent studies suggest its additional use in alopecia topical treatment, generating a demand for novel formulations with specific delivery into hair follicles. Hence, a selective analytical method for drug quantification in follicular structures and skin layers is required. For this, a simple HPLC‐UV method was developed. Quantification was performed using a RP‐C₁₈ column (4.6 mm × 15 cm, 5 μm), with a mixture of methanol–acetonitrile–water (50:15:35 v /v) as mobile phase, a flow rate of 1.2 mL/min, oven temperature of 30°C, injection volume of 50 μL and detection at 240 nm. The optimized conditions enabled a 12 min running with CLO elution at 10.1 min and resolution of 2.424 from skin matrix interferences. Validation was performed in accordance with International Conference on Harmonization guidelines and fulfilled the criteria of selectivity, linearity (0.5–15.0 μg/mL), robustness, precision, accuracy and limits of detection and quantification (0.02 and 0.07 μg/mL, respectively). The validated method was successfully applied for CLO quantification following in vitro skin permeation experiments and differential tape‐stripping for hair follicle deposition determination, demonstrating its suitability.     

Versatile chromatographic method for catechin determination in development of topical formulations containing natural extracts

Catechin is found in several natural sources, as Eugenia dysenterica and Syzygium cumini extracts. Its antioxidant and UV‐protective properties suggest a potential use in cosmetic and dermatological formulations. A simple analytical method capable of giving support to experiments performed along the development of topical formulations containing this natural substance (i.e. drug assay, skin permeation and stability studies), however, is still needed. Thus, this work aimed to develop and validate a selective HPLC method for catechin determination during the development of topical formulations. Separation was achieved using an RP‐C₁₈ column (300 × 3.9 mm; 10 μm), with a mobile phase of methanol–phosphoric acid 0.01 m (15: 85, v/v), a flow rate of 0.8 mL/min, temperature set at 40°C and UV detection at 230 nm. The method was linear in a range from 0.5 to 10.0 μg/mL (r = 0.9998), precise with an overall variation coefficient of 5.5% and accurate with catechin recovery from the skin layers >85%. Additionally, the method was sensitive (limit of detection, 0.109 μg/mL; limit of quantification, 0.342 μg/mL) and selective against plant extracts, skin matrices and formulation interferents, as well as catechin degradation products. It was also robust regarding both methodology parameters and analytical stability.     

An HPLC‐UV method for determining plasma dimethylacetamide concentrations in patients receiving intravenous busulfan

Dimethylacetamide (DMA) is a solvent used in the preparation of intravenous busulfan, an alkylating agent used in blood or marrow transplantation. DMA may contribute to hepatic toxicity, so it is important to monitor its clearance. The aim of this study was to develop an HPLC‐UV assay for measurement of DMA in human plasma. After precipitation of plasma proteins with acetonitrile followed by dilution (1:4) with water, the extract was injected onto the HPLC and detected at 195 nm. Separation was performed using a Cogent‐HPS 5 μm C₁₈ column (250 × 4.6 mm) preceded by a Brownlee 7 μm RP₁₈, pre‐column (1.5 cm × 3.2 mm). The mobile phase was 25 mm sodium phosphate buffer (pH 3), containing 2.5% (v /v) acetonitrile and 0.0005% (v /v) sodium‐octyl‐sulfonate. Using a flow rate of 1 mL/min, the retention times of DMA and the internal standard (IS), 2‐chloroacetamide, were 9.5 and 3.5 min, respectively. Peak area ratio (DMA:IS) was a linear function of concentration from 1 to 1000 μg/mL. There was excellent intraday precision (<5% for 5–700 μg/mL DMA), accuracy (<3% deviation from the true concentration) and recovery (74–98%). The limits of detection and quantification were 1 and 5 μg/mL, respectively. In eight children who received intravenous busulfan, DMA concentrations ranged from 110 to 438 μg/mL.     

Development and validation of a HPLC method for determination of levonorgestrel and quinestrol in rat plasma

Levonorgestrel and quinestrol, commonly known as EP‐1, has long been used in the control of wild rodents. Up to the present time, however, no method for simultaneous quantification of levonorgestrel and quinestrol in rat plasma has been reported. In the present study, a sensitive reverse‐phase high‐performance liquid chromatography with ultraviolet detection (RP‐HPLC‐UV) method for quantification of levonorgestrel and quinestrol in rat plasma has been developed. It uses a Kromasil ODS C₁₈ column and acetonitrile‐0.1% formic acid (85 : 15, v/v) mobile phase at ambient temperature. The plasma sample was prepared by hexane–isoamyl alcohol extraction (90 : 10, v/v). The flow rate and detection wavelength were 1.0 mL/min and 230 nm. The correlation coefficients were greater than 0.9995 within 0.08–50 μg/mL for levonorgestrel and 0.12–50 μg/mL for quinestrol, and the limits of detection were 0.02 and 0.05 μg/mL for levonorgestrel and quinestrol, respectively. Average recovery ranged from 92.5 to 96.3% and inter‐day RSDs were less than 7.56%. This method can be applied to the further pharmacokinetic study of levonorgestrel and quinestrol in rat plasma. Copyright © 2009 John Wiley & Sons, Ltd.     

A simple RP‐HPLC method for quantification of columbianadin in rat plasma and its application to pharmacokinetic study

A rapid and sensitive reversed‐phase high‐performance liquid chromatographic (RP‐HPLC) method was developed to investigate pharmacokinetics of columbianadin, one of the main bioactive constituents in the roots of Angelica pubescens f. biserrata, in rat plasma after intravenous administration to rats at two doses of 10 and 20 mg/kg. The method involves a plasma clean‐up step using liquid–liquid extraction by diethyl ether, followed by RP‐HPLC separation and detection. Separation of columbianadin was performed on an analytical Diamonsil? ODS C₁₈ column, with a mobile phase of MeOH–H₂O (85 : 15, v/v) at a flow‐rate of 1.0 mL/min, and UV detection was set at 325 nm. The retention time of columbianadin and scoparone (internal standard) was 6.7 and 3.5 min, respectively. The calibration curve was linear over the range of 0.2–20.0 μg/mL (r² = 0.9986) in rat plasma. The lower limits of detection and quantification were 0.05 and 0.1 μg/mL, respectively. The extraction recovery from plasma was in the range of 81.61–89.93%. The intra‐ and inter‐day precisions (relative standard deviation) were between 1.01 and 9.33%, with accuracies ranging from 89.76 to 109.22%. The results indicated that the method established was suitable for the determination and pharmacokinetic study of columbianadin in rat plasma. Copyright © 2009 John Wiley & Sons, Ltd.     

Celecoxib determination in different layers of skin by a newly developed and validated HPLC‐UV method

A simple, rapid and sensitive analytical procedure for the measurement of celecoxib (CXB) levels in skin samples after in vitro penetration studies was developed and validated. In vitro permeability studies in porcine skin were performed for quantification of CXB at different layers of skin, the stratum corneum (SC) and epidermis plus dermis (EP + D) as well as in the acceptor solution (AS) to assess CXB permeation through skin. CXB was quantified by HPLC using a C₁₈ column and UV detection at 251 nm. The mobile phase was methanol–water 72:28 (v/v) and the flow‐rate was 0.8 mL/min. The CXB retention time was 5 min. The assay was linear for CBX in the concentration range of 0.1–3.0 μg/mL in the AS (drug permeated through skin) and 5.0–50.0 μg/mL for drug retained in SC and [EP + D] in vitro. The linear correlation coefficients for the different calibration curves were equal or greater than 0.99. Intra‐ and inter‐assay variabilities were below 8.0%. Extraction of CXB from skin samples showed recoveries higher than 95.0% after 15 min of ultrasonic sound and centrifugation at 2500 rpm for 3 min. The method was considered appropriate for the assay of CXB in skin samples, after in vitro cutaneous penetration studies. Copyright © 2011 John Wiley & Sons, Ltd.     

Simultaneous Determination of Ceftriaxone Sodium and Non Steroidal Anti‐Inflammatory Drugs in Pharmaceutical Formulations and Human Serum by RP‐HPLC

An accurate, sensitive and least time consuming reverse phase high performance liquid chromatographic (RP‐HPLC) method for the estimation of ceftriaxone in the presence of non steroidal anti‐inflammatory drugs in formulation and human serum has been developed and validated. Chromatographic separation was conducted on prepacked Purospher Star, C18 (5 μm, 250 × 4.6 mm) column at room temperature using methanol:water:acetonitrile (80:15:5 v/v/v) as a mobile phase, pH adjusted at 2.8 with ortho‐phosphoric acid and at a flow rate of 1.0 mL/minute, while UV detection was performed at 270 nm. The results obtained showed a good agreement with the declared content. The method shows good linearity in the range of 2.5‐25 μg/mL ceftriaxone serum concentrations with a correlation coefficient 0.999 (inter‐ and intra‐day RSD < 2.0%). The limit of detection and quantification for ceftriaxone and NSAID's in pharmaceutical formulation and serum were in the range 0.51‐1.54 μg/mL. Analytical recovery was >98.1%. The proposed method may be used for the quantitative analysis of commonly administered non steroidal anti‐inflammatory drugs i.e. tiaprofenic acid, naproxen sodium, flurbiprofen, diclofenac acid and mefenamic acid alone or in combination with ceftriaxone from raw materials, dosage formulations and in serum. The established HPLC method is rapid, accurate and selective, because of its sensitivity and reproducibility.     

Chiral separation and thermodynamic investigation of WCK 3023: A novel oxazolidinone antibacterial agent,application to pre‐clinical pharmacokinetic study

A chiral liquid chromatographic method was developed and validated for the quantification of R‐enantiomer impurity (RE) in WCK 3023 (S‐enantiomer), a new drug substance. The separation was achieved on Chiralpak IA (amylose‐based immobilized chiral stationary phase), using a mobile phase consisting of n‐hexane–ethanol–trifluoroacetic acid (70:30:0.2, v/v/v) at a flow rate of 1.0 mL/min. The method was extensively validated for the quantification of RE in WCK 3023 and proved to be robust. For RE the detector response was linear over the concentration range of 0.11–5 μg/mL. The limit of quantitation and limit of detection for RE were 0.11 and 0.04 μg/mL respectively. Average recovery of the RE was in the range of 98.11–99.55%. The developed method was specific, sensitive, precise and accurate for quantitative determination of RE in WCK 3023. The impact of thermodynamic parameters on the chiral separation was evaluated. The method was employed for controlling the enantiomeric impurity in the lots of WCK 3023 used for pre‐clinical studies. The method was successfully applied to evaluate the possible conversion of WCK 3023 to RE in rat serum samples during pre‐clinical pharmacokinetic studies.     

Optimization of dispersive liquid–liquid microextraction with central composite design for preconcentration of chlordiazepoxide drug and its determination by HPLC‐UV

A simple, rapid, and sensitive method based on dispersive liquid–liquid microextraction combined with HPLC‐UV detection applied for the quantification of chlordiazepoxide in some real samples. The effect of different extraction conditions on the extraction efficiency of the chlordiazepoxide drug was investigated and optimized using central composite design as a conventional efficient tool. Optimum extraction condition values of variables were set as 210 μL chloroform, 1.8 mL methanol, 1.0 min extraction time, 5.0 min centrifugation at 5000 rpm min^?1, neutral pH, 7.0% w/v NaCl. The separation was reached in less than 8.0 min using a C₁₈ column using isocratic binary mobile phase (acetonitrile/water (60:40, v/v)) with flow rate of 1.0 mL min^?1. The linear response (r² > 0.998) was achieved in the range of 0.005–10 μg mL^?1 with detection limit 0.0005 μg mL^?1. The applicability of this method for simultaneous extraction and determination of chlordiazepoxide in four different matrices (water, urine, plasma, and chlordiazepoxide tablet) were investigated using standard addition method. Average recoveries at two spiking levels were over the range of 91.3–102.5% with RSD < 5.0% (n = 3). The obtained results show that dispersive liquid–liquid microextraction combined with HPLC‐UV is a fast and simple method for the determination of chlordiazepoxide in real samples.     

HPLC‐DAD method for the simultaneous determination of zofenopril and hydrochlorothiazide in oral pharmaceutical formulations

An HPLC method with DAD detection was developed and validated for the simultaneous determination of zofenopril and hydrochlorothiazide in tablets. The separation was carried out through a gradient elution using an Agilent LiChrospher C18 column (250×4.0 mm id, 5 μm) and a mobile phase consisting of (A) water–TFA (99.9:0.1 v/v) and (B) acetonitrile–TFA (99.1:0.1 v/v) delivered at a flow‐rate of 1.0 mL/min. 8‐Chlorotheophylline was used as internal standard. Calibration curves were found to be linear for the two drugs over the concentration ranges of 5.0–40 and 1.0–20 μg/mL for zofenopril and hydrochlorothiazide, respectively. Linearity, precision, accuracy, specificity and robustness were determined in order to validate the proposed method, which was further applied to the analysis of commercial tablets. The proposed method is simple and rapid, and gives accurate and precise results.     

HPLC‐ESI‐MS/MS analysis and pharmacokinetics of luteoloside,a potential anticarcinogenic component isolated from Lonicera japonica,in beagle dogs

Luteoloside is a potential anticarcinogenic component isolated from Lonicera japonica, a traditional Chinese medicine (TCM). This study details the development and validation of a sensitive and accurate HPLC‐ESI‐MS/MS method for the quantification of luteoloside in dog plasma. Sample pretreatment includes simple protein precipitation using methanol–acetonitrile (1:1, v/v). A Phenomenex Gemini C₁₈ column (2.0 × 50 mm, i.d., 3.5 µm) was used to separate luteoloside and internal standard by gradient mode with mobile phase consisting of water containing 0.1% formic acid and methanol containing 0.1% formic acid at a flow rate of 0.40 mL/min with a column temperature of 25°C. The detection was performed by positive ion electrospray ionization (ESI) in multiple reaction monitoring mode. The calibration curves were linear (R > 0.995) over the concentration range 1.0–2000 ng/mL and the lower limit of quantification was 1.0 ng/mL. The intra‐day and inter‐day precisions (RSD) were all <15%, accuracies (RE) were within the range of ±15%, and recoveries were between 85.0 and 115%. The validated HPLC‐ESI‐MS/MS method was successfully applied to determine plasma concentrations of luteoloside after intravenous administration of luteoloside at a dose level of 20 mg/kg. Copyright © 2012 John Wiley & Sons, Ltd.     

Simple HPLC‐UV for the quantification of a new leishmanicidal candidate (E)‐1‐4(trifluoromethyl) benzylidene)‐5‐(2‐4‐dichlorozoyl) carbonylhydrazine (LASSBio‐1736) in rat plasma for pharmacokinetics assessment

In this study, a sensitive HPLC‐UV assay was developed and validated for the determination of LASSBio‐1736 in rat plasma with sodium diclofenac as internal standard (IS). Liquid–liquid extraction using acetonitrile was employed to extract LASSBio‐1736 and IS from 100 μL of plasma previously basified with NaOH 0.1 M. Chromatographic separation was carried on Waters Spherisorb^®S5 ODS2 C₁₈ column (150 × 4.6 mm, 5 μm) using an isocratic mobile phase composed by water with triethylamine 0.3% (pH 4), methanol and acetonitrile grade (45:15:40, v/v/v) at a flow rate of 1 mL/min. Both LASSBio‐1736 and IS were eluted at 4.2 and 5 min, respectively, with a total run time of 8 min only. The lower limit of quantification was 0.2 μg/mL and linearity between 0.2 and 4 μg/mL was obtained, with an R² > 0.99. The accuracy of the method was >90.5%. The relative standard deviations intra and interday were <6.19 and <7.83%, respectively. The method showed the sensitivity, linearity, precision, accuracy and selectivity required to quantify LASSBio‐1736 in preclinical pharmacokinetic studies according to the criteria established by the US Food and Drug Administration and European Medicines Agency. Copyright © 2016 John Wiley & Sons, Ltd.     

Development and validation of bioanalytical method for quantification of cycloserine in human plasma by liquid chromatography–tandem mass spectrometry: Application to pharmacokinetic study

A selective, sensitive and high‐throughput liquid chromatography–tandem mass spectrometry bioanalytical method has been developed for the estimation of cycloserine in human plasma, employing cytosine as the internal standard. The extraction of the analyte was facilitated by solid‐phase extraction using 100 μL of human plasma. The separation was carried out on a BDS Hypersil C₁₈ (150 × 4.6 mm, 5 μm) column using a mixture of 0.2% formic acid in HPLC‐grade water, methanol and acetonitrile (70:15:15, v/v/v) as mobile phase at a flow rate of 1.0 mL/min. The method was linear over the range of 0.20–20 μg/mL with r² > 0.99. Complete validation of the method was performed as per US Food and Drug Administration guidelines and the results met acceptance criteria. Applying the present method, the clinical pharmacokinetics of cycloserine following oral administration of 250 mg cycloserine was studied under fasting conditions. Assay reproducibility was also verified by incurred sample reanalysis.     

Simultaneous quantification of imatinib and its main metabolite N‐demethyl‐imatinib in human plasma by liquid chromatography–tandem mass spectrometry and its application to therapeutic drug monitoring in patients with gastrointestinal stromal tumor

The aim of this study was to improve and validate a more stable and less time‐consuming method based on liquid chromatography and tandem mass spectrometry (LC‐ MS/MS) for the quantitative measurement of imatinib and its metabolite N‐ demethyl‐imatinib (NDI) in human plasma. Separation of analytes was performed on a Waters XTerra RP₁₈ column (50 × 2.1 mm i.d., 3.5 μm) with a mobile phase consisting of methanol–acetonitrile–water (65:20:15, v /v/v) with 0.05% formic acid at a flow‐rate of 0.2 mL/min. The Quattro MicroTM triple quadruple mass spectrometer was operated in the multiple‐reaction‐monitoring mode via positive electrospray ionization interface using the transitions m /z 494.0 → 394.0 for imatinib, m /z 479.6 → 394.0 for NDI and m /z 488.2 → 394.0 for IS. The method was linear over 0.01–10 μg/mL for imatinib and NDI. The intra‐ and inter‐day precisions were all <15% in terms of relative standard deviation, and the accuracy was within ±15% in terms of relative error for both imatinib and NDI. The lower limit of quantification was identifiable and reproducible at 10 ng/mL. The method was sensitive, specific and less time‐consuming and it was successfully applied in gastrointestinal stromal tumor patients treated with imatinib.     

Simultaneous determination of cucurbitacin IIa and cucurbitacin IIb of Hemsleya amabilis by HPLC–MS/MS and their pharmacokinetic study in normal and indomethacin‐induced rats

A selective and sensitive HPLC–MS/MS method was developed for the simultaneous determination of cucurbitacin IIa (cuIIa) and cucurbitacin IIb (cuIIb), the major bioactive cucurbitacins of Hemsleya amabilis, in rat plasma using euphadienol as internal standard (IS). After liquid–liquid extraction with dichloromethane, separation was achieved on a Syncronis HPLC C₁₈ column (150 mm × 4.6 mm, 5 μm) using an isocratic mobile phase system consisting of acetonitrile–water (85:15, v/v) at a flow rate of 0.6 mL/min with a split ratio of 1:2. Detection was performed on a TSQ Quantum Ultra mass spectrometer equipped with an positive‐ion electrospray ionization source. The lower limits of quantification (LLOQs) were 0.25 and 0.15 ng/mL for cuIIa and cuIIb, respectively. The intra‐ and inter‐day precision was <11.5% for the LLOQs and each quality control level of the analytes, and accuracy was between ?9.1 and 7.6%. The extraction recoveries of the analytes and IS from rat plasma were all >87.1%. The method was fully validated and applied to compare the pharmacokinetic profiles of the two cucurbitacins in rat plasma after oral administration of H. amabilis extract between normal and indomethacin‐induced rats. Copyright © 2016 John Wiley & Sons, Ltd.     

Development and validation of a high‐performance liquid chromatographic method for the determination of stemoninine in rat plasma after administration of Stemona tuberosa extracts

For the first time, an HPLC method was developed and validated for the determination of stemoninine in plasma after oral and intravenous administration of the extract of the roots of Stemona tuberosa to rats. Plasma samples were analyzed on a Waters reversed‐phase C₁₈ column using a gradient mobile‐phase of eluent A (water containing 0.1% formic acid and 0.2% triethylamine, pH 3.68) and eluent B (acetonitrile–water, 50:50, v/v). The flow rate was 1.0 mL/min and the detector wavelength was 210 nm. The Waters Oasis solid‐phase extraction cartridge was applied for the preparation of plasma samples with high recovery. A good linear relationship was obtained in the concentration range of 1.55–124 µg/mL (r = 0.9995). The limits of quantification and detection were 1.55 and 0.42 µg/mL, respectively. The average recoveries ranged from 91.11 to 96.43% in plasma at stemoninine concentrations of 3.10, 62.0 and 99.2 µg/mL. Intra‐ and inter‐batch coefficient of variations were 3.27–5.37% and 2.49–3.92%, respectively. This method was successfully applied to pharmacokinetic studies after oral and intravenous administration of Stemona tuberosa extract in rats. Copyright © 2010 John Wiley & Sons, Ltd.     

Quantification of 1‐(propan‐2‐ylamino)‐4‐propoxy‐9H‐thioxanthen‐9‐one (TX5), a newly synthetized P‐glycoprotein inducer/activator,in biological samples: method development and validation

A simple, rapid and economical method was developed and validated for the analysis and quantification of 1‐(propan‐2‐ylamino)‐4‐propoxy‐9H ‐thioxanthen‐9‐one (TX5), a P‐glycoprotein inducer/activator, in biological samples, using reverse‐phase high‐performance liquid chromatography (HPLC). A C₁₈ column and a mobile phase composed of methanol–water (90/10, v /v) with 1% (v/v) triethylamine, at a flow rate of 1 mL/min, were used for chromatographic separation. TX5 standards (0.5–150 μm ) were prepared in human serum. Methanol was used for TX5 extraction and serum protein precipitation. After filtration, samples were injected into the HPLC apparatus and TX5 was quantified by a conventional UV detector at 255 nm. The TX5 retention time was 13 min in this isocratic system. The method was validated according to ICH guidelines for specificity/selectivity, linearity, accuracy, precision, limits of detection and quantification (LOD and LOQ) and recovery. The method was proved to be selective, as there were no interferences of endogenous compounds with the same retention time of TX5. Also, the developed method was linear (r ² ≥ 0.99) for TX5 concentrations between 0.5 and 150 μm and the LOD and LOQ were 0.08 and 0.23 μm , respectively. The results indicated that the reported method could meet the requirements for TX5 analysis in the trace amounts expected to be present in biological samples.