Simultaneous HPTLC and RP-HPLC methods for determination of bumadizone in the presence of its alkaline-induced degradation product

Accurate, selective, sensitive and precise HPTLC‐densitometric and RP‐HPLC methods were developed and validated for determination of bumadizone calcium semi‐hydrate in the presence of its alkaline‐induced degradation product and in pharmaceutical formulation. Method A uses HPTLC‐densitometry, depending on separation and quantitation of bumadizone and its alkaline‐induced degradation product on TLC silica gel 60 F₂₅₄ plates, using hexane–ethyl acetate–glacial acetic acid (8:2:0.2, v/v/v) as a mobile phase followed by densitometric measurement of the bands at 240 nm. Method B comprises RP‐HPLC separation of bumadizone and its alkaline‐induced degradation product using a mobile phase consisting of methanol–water–acetonitrile (20:30:50, v/v/v) on a Phenomenex C₁₈ column at a flow‐rate of 2 mL/min and UV detection at 235 nm. The proposed methods were successfully applied to the analysis of bumadizone either in bulk powder or in pharmaceutical formulation without interference from other dosage form additives, and the results were statistically compared with the established method. Copyright © 2011 John Wiley & Sons, Ltd.     

A rapid and sensitive LC–MS/MS method for quantification of quercetin‐3‐O‐β‐d‐glucopyranosyl‐7‐O‐β‐d‐gentiobioside in plasma and its application to a pharmacokinetic study

A rapid and sensitive LC–MS/MS method with good accuracy and precision was developed and validated for the pharmacokinetic study of quercetin‐3‐O‐β‐d ‐glucopyranosyl‐7‐O‐β‐d ‐gentiobioside (QGG) in Sprague–Dawley rats. Plasma samples were simply precipitated by methanol and then analyzed by LC–MS/MS. A Venusil® ASB C₁₈ column (2.1 × 50 mm, i.d. 5 μm) was used for separation, with methanol–water (50:50, v/v) as the mobile phase at a flow rate of 300 μL/min. The optimized mass transition ion‐pairs (m/z) for quantitation were 787.3/301.3 for QGG, and 725.3/293.3 for internal standard. The linear range was 7.32–1830 ng/mL with an average correlation coefficient of 0.9992, and the limit of quantification was 7.32 ng/mL. The intra‐ and inter‐day precision and accuracy were less than ±15%. At low, medium and high quality control concentrations, the recovery and matrix effect of the analyte and IS were in the range of 89.06–92.43 and 88.58–97.62%, respectively. The method was applied for the pharmacokinetic study of QGG in Sprague–Dawley rats. Copyright © 2016 John Wiley & Sons, Ltd.     

An LC–MS/MS assay for the quantitative determination of 2‐pyridyl acetic acid,a major metabolite and key surrogate for betahistine,using low‐volume human K2EDTA plasma

Betahistine is widely used for the treatment of vertigo. Owing to first‐pass metabolism, 2‐pyridyl acetic acid (2PAA, major metabolite of betahistine) was considered as surrogate for quantitation. A specific and sensitive LC–MS/MS method was developed and validated for quantitation of 2PAA using turbo‐ion spray in a positive ion mode. A solid‐phase extraction was employed for the extraction of 2PAA and 2PAA d₆ (IS) from human plasma. Chromatographic separation of analytes was achieved using an ACE CN, 5 μm (50 × 4.6 mm) column with a gradient mobile phase comprising acetonitrile–methanol (90:10% v /v) and 0.7% v/v formic acid in 0.5 mm ammonium trifluoroacetate in purified water (100% v/v). The retention times of 1.15 and 1.17 min for 2PAA and internal standard, respectively, were achieved. Quantitation of 2PAA and internal standard was achieved by monitoring multiple reaction monitoring transition pairs (m /z 138.1 to m /z 92.0 and m /z 142.1 to m /z 96.1, respectively). The developed method was validated for various parameters. The calibration curves of 2PAA showed linearity from 5.0 to 1500 ng/mL, with a lower limit of quantitation of 5.0 ng/mL. The bias and precision for inter‐ and intra‐batch assays were <10%. The developed method was used to support clinical sample analysis.     

Simultaneous densitometric determination of shikonin,acetylshikonin, and β‐acetoxyisovaleryl‐shikonin in ultrasonic‐assisted extracts of four Arnebia species using reversed‐phase thin layer chromatography

A simple, precise, and rapid high‐performance thin‐layer chromatographic (HPTLC) method for the simultaneous quantification of pharmacologically important naphthoquinone shikonin ( 1 ) together with its derivatives acetylshikonin ( 2 ), and β‐acetoxyisovalerylshikonin ( 3 ) in four species of genus Arnebia (A. euchroma, A. guttata, A. benthamii, and A. hispidissima) from the Indian subcontinent has been developed. In addition, the effect of solvents with varying polarity (hexane, chloroform, ethyl acetate, and methanol) for the extraction of these compounds was studied. HPTLC was performed on precoated RP‐18 F_254S TLC plates. For achieving good separation, mobile phase consisting of ACN/methanol/5% formic acid in water (40:02:08 v/v/v) was used. The densitometric determination of shikonin derivatives was carried out at 520 nm in reflection/absorption mode. The method was validated in terms of linearity, accuracy, precision, robustness, and specificity. The calibration curves were linear in the range of 100–600 ng for shikonin and acetylshikonin, and 100–1800 ng for β‐acetoxyisovalerylshikonin. Lower LOD obtained for compounds 1 – 3 were 18, 15, and 12 ng, respectively, while the LOQ obtained were 60, 45, and 40 ng, respectively.     

A high‐throughput LC–MS/MS method for determination of flunarizine in human plasma: Pharmacokinetic study with different doses

A high‐throughput and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method has been developed and validated for the determination of flunarizine in human plasma. Liquid–liquid extraction under acidic conditions was used to extract flunarizine and flunarizine‐d8 from 100 μL human plasma. The mean extraction recovery obtained for flunarizine was 98.85% without compromising the sensitivity of the method. The chromatographic separation was performed on Hypersil Gold C₁₈ (50 × 2.1 mm, 3 μm) column using methanol–10 mm ammonium formate, pH 3.0 (90:10, v/v) as the mobile phase. A tandem mass spectrometer (API‐5500) equipped with an electrospray ionization source in the positive ion mode was used for detection of flunarizine. Multiple reaction monitoring was selected for quantitation using the transitions, m/z 405.2 → 203.2 for flunarizine and m/z 413.1 → 203.2 for flunarizine‐d8. The validated concentration range was established from 0.10 to 100 ng/mL. The accuracy (96.1–103.1%), intra‐batch and inter‐batch precision (CV ≤ 5.2%) were satisfactory and the drug was stable in human plasma under all tested conditions. The method was used to evaluate the pharmacokinetics of 5 and 10 mg flunarizine tablet formulation in 24 healthy subjects. The pharmacokinetic parameters C_max and AUC were dose‐proportional.     

Determination of polychlorinated biphenyls by liquid chromatography–atmospheric pressure photoionization–mass spectrometry

This study presents the atmospheric pressure photoionization (APPI) of high‐chlorinated (five or more chlorine atoms) polychlorinated biphenyls (PCBs) using toluene as dopant, after liquid chromatographic separation. Mass spectra of PCB 101, 118, 138, 153, 180, 199, 206 and 209 were recorded by using liquid chromatography‐APPI‐tandem mass spectrometry (LC‐APPI‐MS/MS) in negative ion full scan mode. Intense peaks appeared at m/z that correspond to [M ? Cl + O]^? ions, where M is the analyte molecule. Furthermore, a detailed strategy, which includes designs of experiments, for the development and optimization of LC‐APPI‐MS/MS methods is described. Following this strategy, a sensitive and accurate method with low instrumental limits of detection, ranging from 0.29 pg for PCB 209 to 8.3 pg for PCB 101 on column, was developed. For the separation of the analytes, a Waters XSELECT HSS T3 (100 mm × 2.1 mm, 2.5 µm) column was used with methanol/water as elution system. This method was applied for the determination of the above PCBs in water samples (surface water, tap water and treated wastewater). For the extraction of PCBs from water samples, a simple liquid–liquid extraction with dichloromethane was used. Method limits of quantification, ranged from 4.8 ng l^?1, for PCB 199, to 9.4 ng l^?1, for PCB 180, and the recoveries ranged from 73%, for PCB 101, to 96%, for PCB 199. The estimated analytical figures were appropriate for trace analysis of high‐chlorinated PCBs in real samples. Copyright © 2014 John Wiley & Sons, Ltd.     

A degradation study of cefepime hydrochloride in solutions under various stress conditions by TLC–densitometry

A rapid, accurate and sensitive thin‐layer chromatography (TLC) method with densitometric detection has been developed and validated for the determination of cefepime in pharmaceuticals. Chromatographic separation was achieved on a silica gel TLC F₂₅₄ plates with a mobile phase consisting of ethanol–2‐propanol–glacial acetic acid 99.5%–water (4:4:1:3, v/v). Densitometric detection was carried out at wavelength of 266 nm in reflectance/absorbance mode. The validation of the method was found to be satisfactory with high accuracy (from 99.24 to 101.37%) and precision (RSD from 0.06 to 0.36%). Additionally, the stability of cefepime in solution was investigated, including the effect of pH, temperature and incubation time. Favorable retention parameters (R_f, R_s, α) were obtained under the developed conditions, which guaranteed good separation of the studied components. The degradation process of cefepime hydrochloride was described by kinetic and thermodynamic parameters (k, t_0.1, t_0.5 and E_a). Moreover, the chemical properties of degradation products were characterized by the R_f values, absorption spectra, HPLC‐MS/MS and TLC‐densitometry analysis. As the method could effectively separate the active substance from its main degradation product (1‐methylpyrrolidine), it can be employed as a method to indicate the stability of this drug. Copyright © 2014 John Wiley & Sons, Ltd.     

Developing a robust LC–MS/MS method to quantify Zn‐DTPA,a zinc chelate in human plasma and urine

Quantitation of Zn‐DTPA (zinc diethylenetriamene pentaacetate, a metal chelate) in complex biological matrix is extremely challenging on account of its special physiochemical properties. This study aimed to develop a robust and specific liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for determination of Zn‐DTPA in human plasma and urine. The purified samples were separated on Proteonavi (250 × 4.6 mm, 5 μm; Shiseido, Ginza, Tokyo, Japan) and a C₁₈ guard column. The mobile phase consisted of methanol–2 mm ammonium formate (pH 6.3)–ammonia solution (50:50:0.015, v/v/v), flow rate 0.45 mL/min. The linear concentration ranges of the calibration curves for Zn‐DTPA were 1–100 μg/mL in plasma and 10–2000 μg/mL in urine. The intra‐ and inter‐day precisions for quality control (QC) samples were from 1.8 to 14.6% for Zn‐DTPA and the accuracies for QC samples were from −4.8 to 8.2%. This method was fully validated and successfully applied to the quantitation of Zn‐DTPA in plasma and urine samples of a healthy male volunteer after intravenous infusion administration of Zn‐DTPA. The result showed that the concentration of Zn‐DTPA in urine was about 20 times that in plasma, and Zn‐DTPA was completely (94.7%) excreted through urine in human.     

Simultaneous determination of 75 abuse drugs including amphetamines,benzodiazepines, cocaine,opioids, piperazines,zolpidem and metabolites in human hair samples using liquid chromatography–tandem mass spectrometry

A liquid chromatography–tandem mass spectrometric method for the simultaneous determination of 75 abuse drugs and metabolites, including 19 benzodiazepines, 19 amphetamines, two opiates, eight opioids, cocaine, lysergic acid diethylamide, zolpidem, three piperazines and 21 metabolites in human hair samples, was developed and validated. Ten‐milligram hair samples were decontaminated, pulverized using a ball mill, extracted with 1 mL of methanol spiked with 28 deuterated internal standards in an ultrasonic bath for 60 min at 50°C, and purified with Q‐sep dispersive solid‐phase extraction tubes. The purified extracts were evaporated to dryness and the residue was dissolved in 0.1 mL of 10% methanol. The 75 analytes were analyzed on an Acquity HSS T3 column using gradient elution of methanol and 0.1% formic acid and quantified in multiple reaction monitoring mode with positive electrospray ionization. Calibration curves were linear (r ≥ 0.9951) from the lower limit of quantitation (2–200 pg/mg depending on the drug) to 2000 pg/mg. The coefficients of variation and accuracy for intra‐ and inter‐assay analysis at three QC levels were 4.3–12.9% and 89.2–109.1%, respectively. The overall mean recovery ranged from 87.1 to 105.3%. This method was successfully applied to the analysis of 11 forensic hair samples obtained from drug abusers.     

Simultaneous determination of metoprolol and its metabolites, α‐hydroxymetoprolol and O‐desmethylmetoprolol,in human plasma by liquid chromatography with tandem mass spectrometry: Application to the pharmacokinetics of metoprolol associated with CYP2D6 genotypes

A rapid and simple LC with MS/MS method for the simultaneous determination of metoprolol and its two CYP2D6‐derived metabolites, α‐hydroxy‐ and O‐desmethylmetoprolol, in human plasma was established. Metoprolol (MET), its two metabolites, and the internal standard chlorpropamide were extracted from plasma (50 μL) using ethyl acetate. Chromatographic separation was performed on a Luna CN column with an isocratic mobile phase consisting of distilled water and methanol containing 0.1% formic acid (60:40, v/v) at a flow rate of 0.3 mL/min. The total run time was 3.0 min per sample. Mass spectrometric detection was conducted by ESI in positive ion selected‐reaction monitoring mode. The linear ranges of concentration for MET, α‐hydroxymetoprolol, and O‐desmethylmetoprolol were 2–1000, 2–500, and 2–500 ng/mL, respectively, with a lower limit of quantification of 2 ng/mL for all analytes. The coefficient of variation for the assay's precision was ≤ 13.2%, and the accuracy was 89.1–110%. All analytes were stable under various storage and handling conditions and no relevant cross‐talk and matrix effect were observed. Finally, this method was successfully applied to assess the influence of CYP2D6 genotypes on the pharmacokinetics of MET after oral administration of 100 mg to healthy Korean volunteers.     

An LC–MS/MS method for simultaneous determination of 1,5‐dicaffeoylquinic acid and 1‐O‐acetylbritannilactone in rat plasma and its application to a pharmacokinetic study

A selective and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed for the simultaneous quantitative determination of 1,5‐dicaffeoylquinic acid (1,5‐DCQA) and 1‐O‐ acetylbritannilactone (1‐O‐ ABL) in rat plasma. Chromatographic separation was performed on a Zorbax Eclipse XDB‐C₁₈ column using isocratic mobile phase consisting of methanol–water–formic acid (70:30:0.1, v /v/v) at a flow rate of 0.25 mL/min. The detection was achieved using a triple‐quadrupole tandem MS in selected reaction monitoring mode. The calibration curves of all analytes in plasma showed good linearity over the concentration ranges of 0.850–213 ng/mL for 1,5‐DCQA, and 0.520–130 ng/mL for 1‐O‐ ABL, respectively. The extraction recoveries were ≥78.5%, and the matrix effect ranged from 91.4 to 102.7% in all the plasma samples. The method was successfully applied for the pharmacokinetic study of the two active components in the collected plasma following oral administration of Inula britannica extract in rats.     

Identification of metabolites of isopropyl 3‐(3,4‐dihydroxyphenyl)‐2‐hydroxypropanoate in rats by high‐performance liquid chromatography combined with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry

Isopropyl 3‐(3,4‐dihydroxyphenyl)‐2‐hydroxypropanoate (IDHP) is an investigational new drug having the capacity for treating ailments in the cardiovascular and cerebrovascular system. In this work, a rapid and sensitive method using high‐performance liquid chromatography coupled with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry (HPLC‐ESI‐Q‐TOF‐MS) was developed to reveal the metabolic profile of IDHP in rats after oral administration. The method involved pretreatment of the samples by formic acid–methanol solution (v/v, 5:95), chromatographic separation by an Agilent Eclipse XDB‐C₁₈ column (150 × 4.6 mm i.dx., 5 μm) and online identification of the metabolites by Q‐TOF‐MS equipped with electrospray ionizer. A total of 16 metabolites from IDHP, including four phase I metabolites and 12 phase II metabolites, were detected and tentatively identified from rat plasma, urine and feces. Among these metabolites, Danshensu (DSS), a hydrolysis product of IDHP, could be further transformed to 11 metabolites. These results indicated that DSS was the main metabolite of IDHP in rats and the major metabolic pathways of IDHP in vivo were hydrolysis, O‐methylation, sulfation, glucuronidation and reduction. The results also demonstrated that renal route was the main pathway of IDHP clearance in rat. The present study provided valuable information for better understanding the efficacy and safety of IDHP. Copyright © 2015 John Wiley & Sons, Ltd.     

Simultaneous densitometric determination of anthelmintic drug albendazole and its metabolite albendazole sulfoxide by HPTLC in human plasma and pharmaceutical formulations

A new, simple, accurate and precise high‐performance thin‐layer chromatographic method has been developed and validated for simultaneous determination of an anthelmintic drug, albendazole, and its active metabolite albendazole, sulfoxide. Planar chromatographic separation was performed on aluminum‐backed layer of silica gel 60G F₂₅₄ using a mixture of toluene–acetonitrile–glacial acetic acid (7.0:2.9:0.1, v /v/v) as the mobile phase. For quantitation, the separated spots were scanned densitometrically at 225 nm. The retention factors (R _f) obtained under the established conditions were 0.76 ± 0.01 and 0.50 ± 0.01 and the regression plots were linear (r ² ≥ 0.9997) in the concentration ranges 50–350 and 100–700 ng/band for albendazole and albendazole sulfoxide, respectively. The method was validated for linearity, specificity, accuracy (recovery) and precision, repeatability, stability and robustness. The limit of detection and limit of quantitation found were 9.84 and 29.81 ng/band for albendazole and 21.60 and 65.45 ng/band for albendazole sulfoxide, respectively. For plasma samples, solid‐phase extraction of analytes yielded mean extraction recoveries of 87.59 and 87.13% for albendazole and albendazole sulfoxide, respectively. The method was successfully applied for the analysis of albendazole in pharmaceutical formulations with accuracy ≥99.32%.     

Determination of asperosaponin VI in rat plasma by HPLC‐ESI‐MS and its application to preliminary pharmacokinetic studies

Asperosaponin VI (also named akebia saponin D) is a typical bioactive triterpenoid saponin isolated from the rhizome of Dipsacus asper Wall (Dipsacaceae). In this work, a sensitive high‐performance liquid chromatography–electrospray ionization–mass spectrometry (HPLC‐ESI‐MS) assay has been established for determination of asperosaponin VI in rat plasma. With losartan as the internal standard (IS), plasma samples were prepared by protein precipitation with methanol. Chromatographic separation was performed on a C₁₈ column with a mobile phase of 10 mm ammonium acetate buffer containing 0.05% formic acid–methanol (32 : 68, v/v). The analysis was performed on an ESI in the selected ion monitoring mode using target ions at m/z 951.4 for asperosaponin VI and m/z 423.2 for the IS. The calibration curve was linear over the range 3–1000 ng/mL and the lower limit of quantification was 3.0 ng/mL. The intra‐ and inter‐assay variability values were less than 9.5 and 7.8%, respectively. The accuracies determined at the concentrations of 3.0, 100.0, 300.0 and 1000 ng/mL for asperosaponin VI were within ±15.0%. The validated method was successfully applied to a pharmacokinetic study in rats after oral administration of asperosaponin VI. Copyright © 2009 John Wiley & Sons, Ltd.     

Simultaneous determination of niacin and its metabolites—nicotinamide,nicotinuric acid and N‐methyl‐2‐pyridone‐5‐carboxamide—in human plasma by LC‐MS/MS and its application to a human pharmacokinetic study

An LC‐MS/MS method for the simultaneous quantitation of niacin (NA) and its metabolites, i.e. nicotinamide (NAM), nicotinuric acid (NUA) and N‐methyl‐2‐pyridone‐5‐carboxamide (2‐Pyr), in human plasma (1 mL) was developed and validated using nevirapine as an internal standard (IS). Extraction of the NA and its metabolites along with the IS from human plasma was accomplished using a simple liquid–liquid extraction. The chromatographic separation of NA, NAM, NUA, 2‐Pyr and IS was achieved on a Hypersil‐BDS column (150 ¥ 4.6 mm, 5 mm) column using a mobile phase consisting of 0.1% formic acid : acetonitrile (20:80 v/v) at a flow rate of 1 mL/min. The total run time of analysis was 2 min and elution of NA, NAM, NUA, 2‐Pyr and IS occurred at 1.37, 1.46, 1.40, 1.06 and 1.27 min, respectively. A detailed validation of the method was performed as per the FDA guidelines and the standard curves were found to be linear in the range of 100–20000 ng/mL for NA; 10–1600 ng/mL for NUA and NAM and 50–5000 ng/mL for 2‐Pyr with mean correlation coefficient of ≥0.99 for each analyte. The method was sensitive, specific, precise, accurate and suitable for bioequivalence and pharmacokinetic studies. The developed assay method was successfully applied to a pharmacokinetic study in humans. Copyright © 2010 John Wiley & Sons, Ltd.     

Determination of aliskiren in human serum quantities by HPLC–tandem mass spectrometry appropriate for pediatric trials

The orally active direct renin inhibitor aliskiren is approved for the treatment of essential hypertension in adults. Analytical methods utilized in clinical studies on efficacy and safety have not been fully described in the literature but need a large sample volume ranging from 200 to 700 μL, rendering them unsuitable particularly for pediatric applications. In the assay presented only 100 μL of serum is needed for mixed‐mode solid‐phase extraction. The chromatographic separation was performed on Xselect^TM C₁₈ CSH columns with mobile phase consisting of methanol–water–formic acid (75:25:0.005, v/v/v) and a flow rate of 0.4 mL/min. Running in positive electrospray ionization and multiple reaction monitoring the mass spectrometer was set to analyze precursor ion 552.2 m/z [M + H]⁺ to product ion 436.2 m/z during a total run time of 5 min. The method covers a linear calibration range of 0.146–1200 ng/mL. Intra‐run and inter‐run precisions were 0.4–7.2 and 0.6–12.9%. Mean recovery was at least 89%. Selectivity, accuracy and stability results comply with current European Medicines Agency and Food and Drug Administration guidelines. This successfully validated LC‐MS/MS method with a wide linear calibration range requiring small serum amounts is suitable for pharmacokinetic investigations of aliskiren in pediatrics, adults and the elderly. Copyright © 2012 John Wiley & Sons, Ltd.     

Two validated chromatographic determinations of an antifungal drug,its toxic impurities and degradation product: A comparative study

Tolnaftate, a thionoester anti‐fungal drug, was subjected to alkaline hydrolysis to produce methyl(m‐tolyl)carbamic acid and β ‐naphthol (tolnaftate impurity A). N‐Methyl‐m‐toluidine, tolnaftate impurity D, was synthesized and structurally elucidated along with tolnaftate alkaline degradation products using IR, H¹NMR and MS. Two stability‐indicating HPTLC and RP‐HPLC methods were developed and validated, for the first time, for determination of tolnaftate, its alkaline degradation products and toxic impurities in the presence of methyl paraben, as a preservative in Tinea Cure^® cream. The proposed HPTLC method depended on separation of the studied components on TLC silica gel F₂₅₄ plates using hexane–glacial acetic acid (8:2, v/v) as a developing system and scanning wavelength of 230 nm. The proposed RP‐HPLC method was based on separation of the five components on an Eclipse plus C₁₈ column. The mobile phase used was acetonitrile–water containing 1% ammonium formate (40:60, v/v), with a flow rate of 1 mL/min and detection wavelength of 230 nm. The proposed methods allowed the assay of tolnaftate toxic impurities, β ‐naphthol and N‐methyl‐m‐toluidine, down to 2%, allowing tracing of β ‐naphthol that could be absorbed by the skin causing systemic toxic effects, unlike tolnaftate, indicating the high significance of such determination. International Conference on Harmonization guidelines were followed for validation.     

Determination of ospemifene in human plasma by LC–MS/MS and its application to a human pharmacokinetic study

A highly sensitive, specific and rapid liquid chromatography–tandem mass spectrometry (LC–MS/MS) analytical method has been developed and validated for the determination of ospemifene in human plasma using ospemifene‐d₄ as an internal standard. Solid‐phase extraction technique with Phenomenex Strata X‐33 μm polymeric sorbent cartridges (30 mg/1 mL) was used to extract the analytes from the plasma. The chromatographic separation was achieved on Agilent Eclipse XDB‐Phenyl, 4.6 × 75 mm, 3.5 μm column using the mobile phase composition of methanol and 20 mm ammonium formate buffer (90:10, v/v) at a flow rate of 0.9 mL/min. A detailed method validation was performed as per the US Food and Drug Administration guidelines and the calibration curve obtained was linear (r² = 99) over the concentration range 5.02–3025 ng/mL. The API‐4500 MS/MS was operated under multiple reaction monitoring mode during the analysis. The proposed method was successfully applied to a pharmacokinetic study in healthy human volunteers after oral administration of an ospemifene 60 mg tablet under fed conditions.     

Development of an HPTLC‐based diagnostic method for invasive aspergillosis

A rapid, sensitive and specific high‐performance thin‐layer chromatographic (HPTLC) method was developed and validated for determination of gliotoxin in Aspergillus infected immunocompromised patients with invasive aspergillosis (IA). Densitometric analysis of gliotoxin was carried out in the absorbance mode at 254 nm after single‐step extraction with chloroform. The method uses TLC aluminum plates pre‐coated with silica gel 60F‐254 as a stationary phase and toluene–isoamyl alcohol–methanol (10:0.5:0.5, v/v/v) as mobile phase, which gives compact spot of gliotoxin (R_f = 0.51). The calibration curve was linear (r² ≥ 0.994) between peak area and concentration in the tested range of 100–1000 ng spot^?1 with minimum detectable range 0.025 ng μ^?1 of serum sample. The mean ± SD value of slope and intercept of the standard chromatogram of gliotoxin were found to be 523.2 ± 1.555635 and 915.8 ± 30.68843, respectively. The developed method is simple, rapid, precise and less costly than earlier diagnostic methods, and different serum samples can be run on a single TLC plate for comparative analysis. The proposed method can be used to analyze gliotoxin in patient serum for easy, rapid and cost‐effective diagnosis of IA. Copyright © 2009 John Wiley & Sons, Ltd.     

Liquid chromatography–mass spectrometry for analysis of a novel β2‐adrenoceptor agonist trantinterol and its metabolites in beagle dog urine

A liquid chromatography–tandem mass spectrometry method was developed for the identification of metabolites of trantinterol, a novel β₂‐adrenoceptor agonist, in beagle dog urine. The separation of metabolites was performed on a reversed‐phase C₈ column using 0.1% formic acid in water and methanol (70 : 30, v/v) as the mobile phase. The structural information and elemental information of metabolites were acquired by an electrospray ionization tandem mass spectrometer and a quadrupole time‐of‐flight mass spectrometer, respectively. A total of 13 metabolites were detected and characterized on the basis of their tandem MS/MS fragmentation patterns. The accurate masses of nine metabolites were determined and two metabolites were further confirmed by comparing with reference standards. The metabolic pathways of trantinterol in beagle dog are proposed. Copyright © 2009 John Wiley & Sons, Ltd.