Validation of an enantioselective LC–MS/MS method to quantify enantiomers of (±)‐OTX015 in mice plasma: Lack of in vivo inversion of (−)‐OTX015 to its antipode

A highly sensitive, specific and enantioselective assay has been validated for the quantitation of OTX015 enantiomers [(+)‐OTX015 and (−)‐OTX015] in mice plasma on LC–MS/MS‐electrospray ionization as per regulatory guidelines. Protein precipitation was used to extract (±)‐OTX015 enantiomers and internal standard (IS) from mice plasma. The active [(−)‐OTX015] and inactive [(+)‐OTX015] enantiomers were resolved on a Chiralpak‐IA column using an isocratic mobile phase (0.2% ammonia/acetonitrile 20 : 80, v /v) at a flow rate of 1.2 mL/min. The total run time was 6.0 min. (+)‐OTX015, (−)‐OTX015 and IS eluted at 3.34, 4.08 and 4.77 min, respectively. The MS/MS ion transitions monitored were m/z 492 → 383 for OTX015 and m/z 457 → 401 for IS. The standard curves for OTX015 enantiomers were linear (r ² > 0.998) in the concentration range 1.03–1030 ng/mL. The inter‐ and intraday precisions were in the range 2.20–13.3 and 8.03–12.1% and 3.80–14.4 and 8.97–13.6% for (+)‐OTX015 and (−)‐OTX015, respectively. Both the enantiomers were found to be stable in a battery of stability studies. This novel method has been applied to the study of stereoselective oral pharmacokinetics of (−)‐OTX015 and unequivocally demonstrated that (−)‐OTX015 does not undergo chiral inversion to its antipode in vivo in mice.     

A high‐performance liquid chromatographic analysis and preliminary pharmacokinetic characterization of 3′‐hydroxypterostilbene in rats

A high‐performance liquid chromatographic method was developed for the analysis of 3'‐hydroxypterostilbene. This method involves the use of a Luna^® C₁₈ column with ultraviolet detection at 325 nm. The mobile phase consisted of acetonitrile, water and formic acid (50:50:0.01, v/v/v) with a flow rate of 0.8 mL/min. The calibration curves were linear over the range 0.5–100.0 µg/mL. The mean extraction efficiency was between 97.40 and 111.16%. The precision of the assay was 0.196–14.39% (RSD%), and within 15% at the limit of quantitation (0.5 µg/mL). The bias of the assay was <16% and within 15% at the limit of quantitation. This assay was successfully applied to pre‐clinical pharmacokinetic samples from rat urine and serum. Copyright © 2009 John Wiley & Sons, Ltd.     

Development and validation of LC‐MS/MS method for the estimation of β‐hydroxy‐β‐methylbutyrate in rat plasma and its application to pharmacokinetic studies

A simple, sensitive and specific high‐performance liquid chromatography mass spectrometry (LC‐MS/MS) method was developed and validated for the quantification of β‐hydroxy‐β‐methyl butyrate (HMB) in small volumes of rat plasma using warfarin as an internal standard (IS). The API‐4000 LC‐MS/MS was operated under the multiple reaction‐monitoring mode using the electrospray ionization technique. A simple liquid–liquid extraction process was used to extract HMB and IS from rat plasma. The total run time was 3 min and the elution of HMB and IS occurred at 1.48 and 1.75 min respectively; this was achieved with a mobile phase consisting of 0.1% formic acid in a water–acetonitrile mixture (15:85, v/v) at a flow rate of 1.0 mL/min on a Agilent Eclipse XDB C₈ (150 × 4.6, 5 µm) column. The developed method was validated in rat plasma with a lower limit of quantitation of 30.0 ng/mL for HMB. A linear response function was established for the range of concentrations 30–4600 ng/mL (r > 0.998) for HMB. The intra‐ and inter‐day precision values for HMB were acceptable as per Food and Drug Administration guidelines. HMB was stable in the battery of stability studies, viz. bench‐top, autosampler freeze–thaw cycles and long‐term stability for 30 days in plasma. The developed assay method was applied to a bioavailability study in rats. Copyright © 2012 John Wiley & Sons, Ltd.     

On‐line high‐performance liquid chromatography coupled with biochemical detection method for screening of α‐glucosidase inhibitors in green tea

An on‐line high‐performance liquid chromatography–biochemical detection (HPLC‐BCD) method, in which compounds separated by HPLC were on‐line reacted with enzyme and substrate solutions delivered by flow injection and the enzyme inhibition signal was collected by UV detection, was developed to rapidly screen α‐glucosidase inhibitors from green tea extracts in this study. The chromatographic fingerprints and enzyme inhibition profiles of the different brands of green tea could be simultaneously detected by the on‐line HPLC‐BCD method. Enzyme inhibition profiles were detected by the UV detector at 415 nm based on the reaction of α‐glucosidase and p‐nitrophenyl α‐d ‐glucopyranoside (PNPG). PNPG (1.25 mm ), α‐glucosidase (0.4 U/mL) and the flow rate 0.07 mL/min were applied as optimized parameters to detect α‐glucosidase inhibitors in green tea. Four components in green tea showed α‐glucosidase inhibition action and three of them were identified as HHDP‐galloyl glucose, (−)‐epigallocatechin‐3‐gallate and (−)‐epicatechin‐3‐gallate by HPLC–fourier‐transform mass spectrometry (HPLC‐FTMS). Two brands of green tea derived from Mengding and Enshi mountainous areas might be superior to the other samples in the prevention and treatment of diabetes owing to their stronger activities of enzyme inhibitors. The proposed on‐line HPLC‐BCD method could be used to rapidly identify the potential enzyme inhibitors in complex matrixes.     

Determination of 3‐α‐hydroxytibolone in human plasma by LC‐MS/MS: application for a pharmacokinetic study after administration of a tibolone formulation

A new method was developed for the quantitation of 3‐α‐hydroxy tibolone, in human plasma, after oral administration of a tablet formulation containing tibolone (2.5 mg). 3‐α‐Hydroxy tibolone was extracted by a liquid–liquid procedure, using cyproterone acetate as internal standard and chlorobutane as extraction solvent. After extraction, samples were submitted to a derivatization step with p‐toluenesulfonyl isocyanate. A mobile phase consisting of acetonitrile and water (72:28 v/v) was used and chromatographic separation was achieved using Agilent XDB C₁₈ column (100 × 4.6 mm i.d.; 5 µm particle size), at 40°C. Mass spectrometric detection was performed using atmospheric pressure chemical ionization in negative mode for 3‐α‐hydroxy tibolone and in positive mode for cyproterone acetate. The fragmentation transitions were m/z 510.2 → m/z 170.1 and m/z 417.0 → m/z 357.1 for 3‐α‐hydroxy tibolone and cyproterone acetate, respectively. Calibration curves were constructed over the range 100–30,000 pg/mL and the method was shown to be specific, precise and accurate, with a mean recovery rate of 94.2% for 3‐α‐hydroxy tibolone. No matrix effect or carry‐over was detected in the samples. The validated method was applied in a pharmacokinetic study with a tibolone formulation in healthy female volunteers. Copyright © 2013 John Wiley & Sons, Ltd.     

An analytical method for measuring α‐amylase activity in starch‐containing foods

The quality of starch‐containing foods may be significantly impaired by contamination with very small amounts of α‐amylase, which can enzymatically hydrolyze the starch and cause viscosity loss. Thus, for quality control, it is necessary to have an analytical method that can measure low amylase activity. We developed a sensitive analytical method for measuring the activity of α‐amylase (from Bacillus subtilis) in starch‐containing foods. The method consists of six steps: (1) crude extraction of α‐amylase by centrifugation and filtration; (2) α‐amylase purification by desalting and anion‐exchange chromatography; (3) reaction of the purified amylase with boron‐dipyrromethene (BODIPY)‐labeled substrate, which releases a fluorescent fragment upon digestion of the substrate, thus avoiding interference from starch derivatives in the sample; (4) stopping the reaction with acetonitrile; (5) reversed‐phase solid‐phase extraction of the fluorescent substrate to remove contaminating dye and impurities; and (6) separation and measurement of BODIPY fluorescence by HPLC. The proposed method could quantify α‐amylase activities as low as 10 mU/mL, which is enough to reduce the viscosity of starch‐containing foods. Copyright © 2012 John Wiley & Sons, Ltd.     

A novel sorbent based on carbon nanotube/amino‐functionalized sol–gel for the headspace solid‐phase microextraction of α‐bisabolol from medicinal plant samples using experimental design

A novel sol–gel coating on a stainless‐steel fiber was developed for the first time for the headspace solid‐phase microextraction and determination of α‐bisabolol with gas chromatography and flame ionization detection. The parameters influencing the efficiency of solid‐phase microextraction process, such as extraction time and temperature, pH, and ionic strength, were optimized by the experimental design method. Under optimized conditions, the linear range was between 0.0027 and 100 μg/mL. The relative standard deviations determined at 0.01 and 1.0 μg/mL concentration levels (n = 3), respectively, were as follows: intraday relative standard deviations 3.4 and 3.3%; interday relative standard deviations 5.0 and 4.3%; and fiber‐to‐fiber relative standard deviations 6.0 and 3.5%. The relative recovery values were 90.3 and 101.4% at 0.01 and 1.0 μg/mL spiking levels, respectively. The proposed method was successfully applied to various real samples containing α‐bisabolol.     

Enantioselective determination of R‐(−)‐manidipine and S‐(+)‐manidipine in human plasma by a sensitive and selective chiral LC–MS/MS assay

A sensitive and selective liquid chromatography–tandem mass spectrometric (LC–MS/MS) assay method has been developed and validated for the enantioselective determination of manidipine in human plasma using isotope‐labeled compounds as internal standards. After solid‐phase extraction, R ‐(−)‐manidipine and S ‐(+)‐manidipine were chromatographed on a Chiralpack IC‐3 C₁₈ column using a isocratic mobile phase composed of 2 mm ammonium bicarbonate and acetonitrile (15:85, v /v). The precursor ion to product ion transitions for the enantiomers and internal standards were monitored in the multiple reaction monitoring and positive ionization mode using an API‐4000 mass spectrometer. The method was linear over the concentration range of 0.05–10.2 ng/mL for both enantiomers. The precision and accuracy results over five concentration levels in five different batches were well within the acceptance limits. The mean extraction recovery was >80% for both enantiomers. A variety of stability tests were executed in plasma and in neat samples, which complies with the FDA guidelines. After complete validation, the method was successfully applied to a pharmacokinetic study of a manidipine 20 mg oral dose in 10 healthy South India subjects under fasting conditions. The assay reproducibility is shown through incurred samples reanalysis of 20 subject plasma samples.     

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.     

Quantitative determination of pravastatin and its metabolite 3α‐hydroxy pravastatin in plasma and urine of pregnant patients by LC‐MS/MS

This report describes the development and validation of a chromatography/tandem mass spectrometry method for the quantitative determination of pravastatin and its metabolite (3α‐hydroxy pravastatin) in plasma and urine of pregnant patients under treatment with pravastatin, as part of a clinical trial. The method includes a one‐step sample preparation by liquid–liquid extraction. The extraction recovery of the analytes ranged between 93.8 and 99.5% in plasma. The lower limits of quantitation of the analytes in plasma samples were 0.106 ng/mL for pravastatin and 0.105 ng/mL for 3α‐hydroxy pravastatin, while in urine samples they were 19.7 ng/mL for pravastatin and 2.00 ng/mL for 3α‐hydroxy pravastatin. The relative deviation of this method was <10% for intra‐ and interday assays in plasma and urine samples, and the accuracy ranged between 97.2 and 106% in plasma, and between 98.2 and 105% in urine. The method described in this report was successfully utilized for determining the pharmacokinetics of pravastatin in pregnant patients enrolled in a pilot clinical trial for prevention of preeclampsia. Copyright © 2015 John Wiley & Sons, Ltd.     

Sensitive determination of glucose in Dulbecco's modified Eagle medium by high‐performance liquid chromatography with 1‐phenyl‐3‐methyl‐5‐pyrazolone derivatization: application to gluconeogenesis studies

A new pre‐column derivative high‐performance liquid chromatography (HPLC) method for determination of d ‐glucose with 3‐O‐methyl‐d ‐glucose (3‐OMG) as the internal standard was developed and validated in order to study the gluconeogenesis in HepG2 cells. Samples were derivatized with 1‐phenyl‐3‐methy‐5‐pyrazolone at 70°C for 50 min. Glucose and 3‐OMG were extracted by liquid–liquid extraction and separated on a YMC‐Triart C₁₈ column, with a gradient mobile phase composed of acetonitrile and 20 mm ammonium acetate solution containing 0.09% tri‐ethylamine at a flow rate of 1.0 mL/min. The eluate were detected using a UV detector at 250 nm. The assay was linear over the range 0.39–25 μm (R² = 0.9997, n = 5) and the lower limit of quantitation was 0.39 μm (0.070 mg/mL). Intra‐ and inter‐day precision and accuracy were <15% and within ±3%, respectively. After validation, the HPLC method was applied to investigate the gluconeogenesis in Dulbecco's modified Eagle medium (DMEM) cultured HepG2 cells. Glucose concentration was determined to be about 1–2.5 μm in this gluconeogenesis assay. In conclusion, this method has been shown to determine small amounts of glucose in DMEM successfully, with lower limit of quantitation and better sensitivity when compared with common commercial glucose assay kits. Copyright © 2015 John Wiley & Sons, Ltd.     

High‐performance liquid chromatographic enantioseparation of (RS)‐bupropion using isothiocyanate‐based chiral derivatizing reagents

A high‐performance liquid chromatographic (HPLC) method for enantioseparation of bupropion was developed using two isothiocyanate‐based chiral derivatizing reagents, (S)‐1‐(1‐naphthyl) ethyl isothiocyanate, (S)‐NEIT, and (R)‐α‐methyl benzyl isothiocyanate, (R)‐MBIT. The diastereomers synthesized with (S)‐NEIT were enantioseparated by reversed‐phase HPLC using gradient elution with mobile phase containing water and acetonitrile, whereas diastereomers synthesized with (R)‐MBIT were enantioseparated using triethyl amine phosphate buffer and methanol. Derivatization conditions were optimized and the method was validated for accuracy, precision and limit of detection. The limit of detection was found to be 0.040–0.043 µg/mL for each of the diastereomers prepared with (S)‐NEIT. Copyright © 2013 John Wiley & Sons, Ltd.     

RP‐HPLC enantioseparation of β‐adrenolytics using micellar mobile phase without organic solvents

Enantioseparation of a few commonly administered racemic β‐adrenolytics (namely, carvedilol, betaxolol, salbutamol and bisoprolol) has been achieved using a water micellar mobile phase containing surfactants (sodium dodecyl sulphate and Brij‐35) without organic solvents as a new approach in RP‐HPLC. Two chiral derivatizing reagents based on enantiomerically pure (S )‐(−)‐levofloxacin were synthesized using N ‐hydroxysuccinimide and N ‐hydroxybenzotriazole as the activation auxiliaries. Diastereomeric derivatives of the chosen β‐adrenolytics were synthesized under microwave irradiation in a very short reaction time. The (S )‐(−)‐levofloxacin moiety enhanced molar absorbance of the diastereomeric derivatives resulting in very low limit of detection (1.618 and 4.902 ng/mL, respectively, for diastereomeric derivatives of (RS )‐betaxolol and better resolution with lower retention times (for all the analytes), in comparison to literature reports. There was 15–20 times less consumption of mobile phase because of lower retention time.     

Separation and determination of polyurethane amine catalysts in polyether polyols by using UHPLC–Q‐TOF‐MS on a reversed‐phase/cation‐exchange mixed‐mode column

A simple, selective, and accurate ultra‐high performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry method was established and validated for the efficient separation and quantification of polyurethane amine catalysts in polyether polyols. Amine catalysts were primarily separated in polyether polyol‐based sample by solid‐phase extraction, and further baseline separated on a reversed‐phase/cation‐exchange mixed‐mode column (SiELC Primesep™ 200) using 0.1% trifluoroacetic acid/acetonitrile as a mobile phase in gradient elution mode at a flow rate of 0.2 mL/min. High‐resolution quadrupole time‐of‐flight mass spectrometry analysis in electrospray ionization positive mode allowed the identification as N,N′‐bis[3‐(dimethylamino)propyl]urea, N‐[2‐(2‐dimethylaminoethoxy)ethyl]‐N‐methyl‐1,3‐propanediamine, and N,N,N′,N′‐tetramethyldipropylenetriamine. The method was validated and presented good linearity for all the analytes in blank matrices within the concentration range of 0.20–5.0 or 0.1–2.0 μg/mL with the correlation coefficients (R²) ranging from 0.986 to 0.997. Method recovery ranged within 81–105% at all three levels (80, 100, and 120% of the original amount) with relative standard deviations of 1.0–6.2%. The limits of detection were in the range of 0.007–0.051 μg/mL. Good precision was obtained with relative standard deviation below 3.2 and 0.72% for peak area and retention time of three amines, respectively.     

Reversed‐Phase High‐Performance Liquid Chromatographic Separation and Determination of 4‐Amino‐azobenzene‐4′,5‐disulfonic Acid and Related Products in Industrial Wastewater Effluents

A simple and rapid reversed‐phase high‐performance liquid chromatographic method for the separation and determination of 4‐amino‐azobenzene‐4′,5‐disulfonic acid (AABDS) and its process‐related impurities was developed. The separation was achieved on a μ‐Bondapak C₁₈ column using 0.15 M ammonium sulfate‐acetonitrile (55:45) (v/v) as eluent. A UV‐visible spectrophotometric detector fixed at 386 nm was used both for detection and quantitation. The method was used not only for quality assurance but also for process development and wastewater management of AABDS.     

The kinetics of polycondensation of α,α′‐dichloro‐p‐xylene with 4,4′‐isopropylidenediphenol using benzyltriethylammonium chloride as a phase‐transfer catalyst

The phase‐transfer catalyzed polycondensation of α,α′‐dichloro‐p‐xylene with 4,4′‐isopropylidenediphenol was carried out using benzylethylammonium chloride in a two‐phase system of an aqueous alkaline solution and benzene at 60 °C under nitrogen atmosphere. The rate of polycondensation was expressed as the combined terms of quaternary onium cation and 4,4′‐isopropylidenediphenolate anion rather than the feed concentration of catalyst and 4,4′‐isopropylidenediphenol. The measured concentrations of hydroxide and chloride anion in the aqueous solution and α,α′‐dichloro‐p‐xylene in the organic phase were used to obtain the reaction rate constant with the integral method, and to analyze the polycondensation mechanism with a cyclic phase‐transfer initiation step in the heterogeneous liquid–liquid system. © 2000 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 38: 3059–3066, 2000     

HPLC determination of γ‐aminobutyric acid and its analogs in human serum using precolumn fluorescence labeling with 4‐(carbazole‐9‐yl)‐benzyl chloroformate

In this study, a simple analytical method for the determination of γ‐aminobutyric acid, gabapentin, and baclofen by using high‐performance liquid chromatography with fluorescence detection was developed. An amidogen‐reactive fluorescence labeling reagent, 4‐(carbazole‐9‐yl)‐benzyl chloroformate was first used to sensitively label these analytes. The completed labeling of these analytes can be finished rapidly only within 5 min at the room temperature (25°C) to form 4‐(carbazole‐9‐yl)‐benzyl chloroformate labeled fluorescence derivatives. These labeled derivatives expressed strong fluorescence property with the maximum excitation and emission wavelengths of 280 and 380 nm, respectively. The labeled derivatives were analyzed using a reversed‐phase Eclipse SB‐C18 column within 10 min with satisfactory shapes. Excellent linearity (R² > 0.995) for all analytes was achieved with the limits of detection and the limits of quantitation in the range of 0.25?0.35 and 0.70?1.10 μg/L, respectively. The proposed method was used for the simultaneous determination of γ‐aminobutyric acid and its analogs in human serum with satisfactory recoveries in the range of 94.5–97.5%.     

Simultaneous determination of ascaridole,p‐cymene and α‐terpinene in rat plasma after oral administration of Chenopodium ambrosioides L. by GC‐MS

A sensitive and reliable GC‐MS method was developed and validated for the simultaneous determination of ascaridole, p‐cymene and α‐terpinene in rat plasma using naphthalene as internal standard. The plasma samples were extracted with ethyl acetate. Chromatographic separation was carried out on a HP‐5MS capillary analytical column (30 m × 0.25 mm, 0.25 µm) and detection was performed on a quadrupole mass spectrometer detector operated under selected ion monitoring mode. The method showed excellent linearity over the investigated concentration range (r > 0.99) with the limit of quantitation down to 50, 10 and 5 ng/mL for ascaridole, p‐cymene and α‐terpinene, respectively. The intra‐day and inter‐day precisions (RSD) were <11.3%, and the accuracy was between 90.7 and 113.8%. The method was successfully applied to investigate the pharmacokinetics of Chenopodium ambrosioides L. following oral administration to rats. Copyright © 2015 John Wiley & Sons, Ltd.     

An improved ultra‐high‐performance liquid chromatography–tandem mass spectrometric method for the quantitation of dexmedetomidine in small volume of pediatric plasma

Dexmedetomidine (Dex), a highly selective α₂‐adrenergic agonist, is used primarily for the sedation and anxiolysis of adults and children in the intensive care setting. A sensitive and selective assay for Dex in pediatric plasma was developed by employing ultra‐high‐performance liquid chromatography–tandem mass spectrometry with d4‐Dex as an internal standard. Dex was extracted from 0.1 mL of plasma by micro‐elution solid‐phase extraction. Separation was achieved with a Waters XBridge C₁₈ column with a flow rate of 0.3 mL/min using a mobile phase comprising 5 mm ammonium acetate buffer with 0.03% formic acid in water and methanol–acetonitrile (50:50, v/v). The intra‐day precision (coefficient of variation) and accuracy for quality control samples ranged from 1.32 to 8.91% and from 92.8 to 108%, respectively. The inter‐day precision and accuracy ranged from 2.13 to 8.45% and from 97.0 to 104%, respectively. The analytical method showed excellent sensitivity using a small sample volume (0.1 mL) with a lower limit of quantitation of 5 pg/mL. This method is robust and has been successfully employed in a pharmacokinetic study of Dex in neonates and infants postoperative from cardiac surgery.     

On‐line 2D‐LC‐ESI/MS/MS determination of rifaximin in rat serum

A highly sensitive and selective on‐line two‐dimensional reversed‐phase liquid chromatography/electrospray ionization–tandem mass spectrometry (2D‐LC‐ESI/MS/MS) method was developed and validated to determine rifaximin in rat serum by direct injection. The 2D‐LC‐ESI/MS/MS system consisted of a restricted access media column for trapping proteins as the first dimension and a Waters C₁₈ column as second dimension using 0.1% aqueous acetic acid:acetonitrile as mobile phase in a gradient elution mode. Rifampacin was used as an internal standard. The linear dynamic range was 0.5–10 ng/mL (r² > 0.998). Acceptable precision and accuracy were obtained over the calibration range. The assay was successfully used in analysis of rat serum to support pharmacokinetic studies. Copyright © 2009 John Wiley & Sons, Ltd.