Liquid-liquid Extraction Technique Research Articles

Simultaneous determination of six C21 steroids of Xiao‐Ai‐Ping injection in rat plasma by LC‐MS/MS

Xiao-Ai-Ping injection (XAPI) is a traditional Chinese medicine that has been widely used to treat cancer. Modern pharmacological studies have demonstrated that C21 steroids are the main active compounds in XAPI. In this study, a sensitive and specific liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed and validated the first time for simultanenous determination of three isomeric pregnane genins (17β-tenacigenin B, tenacigenin B and tenacigenin A) and their corresponding glycosides (tenacigenoside A, tenacissoside F and marsdenoside I) from XAPI in rat plasma. A simple liquid-liquid extraction technique was used after the addition of dexamethasone acetate as internal standard. The chromatography separation of analytes was achieved on an Agilent Zorbax Eclipse XDB-C18 column (3.5 µm, 150 × 3 mm i.d.) using methanol-water as mobile phase in a gradient elution program. Detection was performed in multiple reaction monitoring mode using electrospray ionization in the negative ion mode. The method showed satisfactory linearity over a concentration range 5.00-2000.00 ng/mL for tenacigenin B, tenacigenin A, marsdenoside I and tenacissoside F (r(2) > 0.99), 10.00-4000.00 ng/mL for 17β-tenacigenin B and tenacigenoside A (r(2) > 0.99). Intra- and inter-day precisions (valued as relative standard deviation) were <9.00% and accuracies (as relative error) in the range -6.31 to 7.23%. Finally, this validated method was successfully applied to the pharmacokinetic study of XAPI after intravenous administration to rats.

Sensitive assay for measurement of volatile borneol, isoborneol, and the metabolite camphor in rat pharmacokinetic study of Borneolum (Bingpian) and Borneolum syntheticum (synthetic Bingpian).

Both Borneolum (Chinese name Bingpian; dextrorotatory borneol) and Borneolum syntheticum (synthetic Bingpian; a mixture of optically inactive borneol and isoborneol) have been used for medicinal purposes in Chinese traditional medicine. The aim of this study was to develop a sensitive assay for measuring volatile ingredients borneol, isoborneol, and their metabolite camphor in pharmacokinetic study of Bingpian. Rat plasma samples were prepared using liquid-liquid microextraction: 70 μL of plasma sample (containing 125 nmol/L naphthalene as the internal standard) was extracted with 35 μL of n-hexane. The resulting n-hexane extract (20 μL) was introduced into a gas chromatography/mass spectrometry system using programmable temperature vaporizing-based large-volume injection. The assay was validated to demonstrate its reliability for the intended use. Using this assay, pharmacokinetic studies of Bingpian, synthetic Bingpian, and Fufang-Danshen tablets (containing synthetic Bingpian) were conducted in rats. The extraction efficiency for the analytes and the internal standard from plasma was almost constant with decrease in n-hexane-to-plasma volume ratio, thus enabling a small volume of extracting solvent to be used for sample preparation, and enhancing the assay sensitivity. The lower quantification limit for measuring borneol, isoborneol, and camphor in plasma was 0.98 nmol/L, which was 33-330 times more sensitive than those reported earlier for Bingpian and synthetic Bingpian. The applicability of the miniaturized liquid-liquid extraction technique could be extended to measure other volatile and nonvolatile medicinal compounds in biomatrices, which can be predicted according to the analytes' octanol/water distribution coefficient (logD) and acid dissociation constant (pKa). This assay is sensitive, accurate and free of matrix effects, and can be applied to pharmacokinetic studies of Bingpian, synthetic Bingpian, and Bingpian-containing herbal products.

Development of dry derivatization and headspace solid-phase microextraction technique for the GC-ECD determination of haloacetic acids in tap water

A simple, fast and efficient liquid-liquid extraction (LLE) technique using headspace solid-phase microextraction (HS-SPME), in conjunction with gas chromatography-electron capture detection (GC-ECD) has been developed for the determination of haloacetic acids (HAAs) in tap water. The analytical procedure involves LLE, evaporation of extraction solvent to dryness, derivatization of HAAs into their methyl esters with acidic methanol, HS-SPME using 100-μm polydimethylsiloxane (PDMS) fiber, and GC-ECD determination. The derivatization process was optimized in dry conditions to achieve maximum sensitivity using the following conditions: esterification for 10 min at 55°C in 50 μL methanol, 30 μL sulphuric acid and 0.1 g anhydrous sodium sulphate. The HS-SPME conditions were also optimized and good sensitivity was obtained at a sampling temperature of 25°C, an absorption time of 10 min and a desorption time of 2 min. The linear calibration curves were observed for the concentration ranging from 0.1 to 200 μg/L with the correlation coefficients (R2) greater than 0.993 and the relative standard deviation (RSD) less than 12%. The method detection limits of all analytes ranging from 0.02 to 0.7 μg/L were obtained. The proposed method is compared directly to standard EPA method 552.2 in drinking water, and significant advantage in terms of selectivity was observed. Finally the optimized procedure was applied to the analysis of HAAs in Bizerte drinking water. The studied HAA were detected in all the water samples and the concentration of total HAA5 ranged from 17.8 to 70.3 μg/L.

Prostaglandin Extraction and Analysis in &lt;em&gt;Caenorhabditis elegans&lt;/em&gt;

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, which are lipid signals derived from polyunsaturated fatty acids (PUFAs)10-14. These signalling molecules are difficult to study because of their low abundance and reactive nature. The characteristic feature of prostaglandins is a cyclopentane ring structure located within the fatty acid backbone. In mammals, prostaglandins can be formed through cyclooxygenase enzyme-dependent and -independent pathways10,15. C. elegans synthesizes a wide array of prostaglandins independent of cyclooxygenases6,16,17. A large class of F-series prostaglandins has been identified, but the study of eicosanoids is at an early stage with ample room for new discoveries. Here we describe a procedure for extracting and analyzing prostaglandins and other eicosanoids. Charged lipids are extracted from mass worm cultures using a liquid-liquid extraction technique and analyzed by liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The inclusion of deuterated analogs of prostaglandins, such as PGF2 α-d4 as an internal standard is recommended for quantitative analysis. Multiple reaction monitoring or MRM can be used to quantify and compare specific prostaglandin types between wild-type and mutant animals. Collision-induced decomposition or MS/MS can be used to obtain information on important structural features. Liquid chromatography mass spectrometry (LC-MS) survey scans of a selected mass range, such as m/z 315-360 can be used to evaluate global changes in prostaglandin levels. We provide examples of all three analyses. These methods will provide researchers with a toolset for discovering novel eicosanoids and delineating their metabolic pathways.

Prostaglandin Extraction and Analysis in &lt;em&gt;Caenorhabditis elegans&lt;/em&gt;

Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids1-9. Prostaglandins are an important class of eicosanoids, which are lipid signals derived from polyunsaturated fatty acids (PUFAs)10-14. These signalling molecules are difficult to study because of their low abundance and reactive nature. The characteristic feature of prostaglandins is a cyclopentane ring structure located within the fatty acid backbone. In mammals, prostaglandins can be formed through cyclooxygenase enzyme-dependent and -independent pathways10,15. C. elegans synthesizes a wide array of prostaglandins independent of cyclooxygenases6,16,17. A large class of F-series prostaglandins has been identified, but the study of eicosanoids is at an early stage with ample room for new discoveries. Here we describe a procedure for extracting and analyzing prostaglandins and other eicosanoids. Charged lipids are extracted from mass worm cultures using a liquid-liquid extraction technique and analyzed by liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The inclusion of deuterated analogs of prostaglandins, such as PGF2 α-d4 as an internal standard is recommended for quantitative analysis. Multiple reaction monitoring or MRM can be used to quantify and compare specific prostaglandin types between wild-type and mutant animals. Collision-induced decomposition or MS/MS can be used to obtain information on important structural features. Liquid chromatography mass spectrometry (LC-MS) survey scans of a selected mass range, such as m/z 315-360 can be used to evaluate global changes in prostaglandin levels. We provide examples of all three analyses. These methods will provide researchers with a toolset for discovering novel eicosanoids and delineating their metabolic pathways.

English

A simple, sensitive and accurate liquid chromatography tandem mass spectrometric (LC/MS/MS) method has been developed and validated for determination of varenicline (VRC) in human plasma and pharmaceutical tablets as a tool for therapeutic drug monitoring. The VRC and internal standard (paracetamol, IS) were extracted by liquid-liquid extraction technique. The separation was achieved on C18 column (150mm &times; 4.6 mm, 5 &mu;m, maintained at 25&deg;C) by isocratic mode at a flow rate of 0.7 ml/min using a mobile phase consisted of a mixture of 5 mM ammonium formate, pH 7.5 (A) and (acetonitrile: methanol, 50:50, v/v) (B) in a ratio of A:B (15:85, v/v) for 10 min. The analytes were monitored by electrospray ionization in positive ion multiple reaction monitoring (MRM) mode. Optimization of MRM mode and chromatogrphic conditions were applied to elmeinate the interference peaks and increse of sensitivity. The method was linear (r2&nbsp;= 0.9998) at concentration range of 20.0 to 500.0 ng/ml with lower limit of detection of 6.0 ng/ml. The method was statistically validated for linearity, accuracy, precision and selectivity following Food and Drug Administration (FDA) guidelines. The mean extraction recovery of VRC from human plasma was 87.06 &plusmn; 2.47%. The reproducibility of the method was reliable with the intra- and inter-day precision was &lt; 5% and average accuracy of 103.54%. The validated method was successfully applied to quantify VRC in human plasma as well as bulk and dosage form in quality control laboratory. &nbsp; Key words:&nbsp;Liquid chromatography tandem mass spectrometric (LC/MS/MS), varenicline, human plasma, dosage form

Quantification of curcumin, demethoxycurcumin, and bisdemethoxycurcumin in rodent brain by UHPLC/ESI‐Q‐TOF‐MS/MS after intra‐nasal administration of curcuminoids loaded PNIPAM nanoparticles

An ultra high performance liquid chromatography-electrospray ionization-synapt mass spectrometric method (UHPLC/ESI-QTOF-MS/MS) for the analysis of curcumin (Cur), demethoxycurcumin (DMC), bisdemethoxycurcumin (BDMC) in Wistar rat brain homogenate was developed and validated. The chromatographic separation was achieved on a Waters ACQUITY UPLC™ BEH C18 (2.1mm × 100 mm; 1.7μm) column using isocratic mobile phase, consisting of acetonitrile: 10mM ammonium formate: formic acid (90:10:0.05v/v/v), at a flow rate of 0.2 ml min(-1) . The transitions occurred at m/z 367.0694/217.0598, 337.0717/173.0910, 307.0760/187.0844 for Cur, DMC, BDMC and m/z 307.0344/229.0677 for the IS (Nimesulide) respectively. The recovery of the analytes from Wistar rat brain homogenate was optimized using liquid-liquid extraction technique (LLE) in (ethyl acetate: chloform) mixture. The total run time was 3.0 min and the elution of Cur, DMC, BDMC occurred at 1.6, 1.75, 1.70 min, and for the IS 1.87 min, respectively. The linear dynamic range was established over the concentration range of 1.00 ng mL(-1) to 1000.0 ng mL(-1) (r(2) ; 0.9909 ± 0.0011, 0.9911 ± 0.003, and 0.9919 ± 0.0013) for Cur, DMC, and BDMC, respectively. The intra and inter-assay accuracy in terms of % CV for Cur, DMC, and BDMC was in the range 0.47-2.20, 0.47-1.65, and0.44-2.70, respectively. The lower limit of detection (LOD) and quantitation (LOQ) for Cur, DMC, and BDMC were 0.46, 0.05, 0.16 ng mL(-1) and 0.153, 0.015, 0.052 ng mL(-1) , respectively. Analytes were stable and the method proved to be accurate (recovery, >85%), specific and was applied to evaluate the Cur, DMC, BDMC loaded PNIPAM NPs as vehicles for nose to brain drug delivery.

Recent advances in flow-based sample pretreatment for the determination of metal species by atomic spectrometry

During the last few years, various flow-based separation/preconcentration methodologies have gained pertinent novel advances and exhibited powerful capability in the field of sample pretreatment and their hyphenation with detection by atomic spectrometry. The present mini-review presents and discusses the progress of flow-based sample processing approaches commonly used for the assay of trace elemental species with detection by atomic spectrometry, including preliminary sample pretreatment, solid phase extraction (including solid phase microextraction), liquid-liquid extraction, vapor generation and dialysis techniques. Special emphasis has been paid on the novel applications and analytical procedures hyphenated with atomic spectrometry. The future perspectives of flow-based sample pretreatment protocols in the determination of trace elements and their speciation are also discussed.

Simultaneous Determination of Oridonin, Ponicidin and Rosmarinic Acid from Herba Isodi Rubescentis Extract by LC–MS-MS in Rat Plasma

An analytical method was developed for the simultaneous quantification of oridonin, ponicidin and rosmarinic acid in rat plasma using liquid chromatography-tandem mass spectrometry with electrospray ionization in positive and negative ion modes of operation. Multiple reaction monitoring was used for quantification using the precursor to product ion pairs of m/z 359.100 → 160.9 (rosmarinic acid), m/z 150.1 → 106.9 (acetaminophen as internal standard 1), m/z 365.1 → 347.3 (oridonin), m/z 363.3 → 345.2 (ponicidin) and m/z 258.3 → 201.0 (dextrorphan as internal standard 2). Optimum chromatographic separation was achieved with a BDS Hypersil C18 column (100 × 2.1 mm, 2.4 µm) and a mobile phase consisting of acetonitrile-5mM ammonium acetate containing 0.1% acetic acid (25:75, v/v). The analytes were extracted from rat plasma using a single liquid-liquid extraction technique and the calibration curve was linear within the concentration range of 2-1,000.0 ng/mL for three analytes. The extraction recovery was above 70%, the intra-day and inter-day precision of the quality control (QC) samples was less than 12.45% and the accuracy of the QC samples was 96.99-105.79% of the nominal values. The method was sensitive and reliable and was successfully applied to a pharmacokinetic and bioavailability study of Herba Isodi Rubescentis extract in rats.

Flow Chemiluminescence Determination of Antimony(III,V) Using a Rhodamine B-Cetyltrimethylammonium Chloride Reversed Micelle System Following Liquid–Liquid Extraction

A flow chemiluminescence (CL) method combined with a liquid-liquid extraction technique is proposed for the indirect determination of antimony in aqueous samples using rhodamine B (RB). In the liquid-liquid extraction process, the antimony(V) chloro-complex anion, [SbCl(6)](-), was extracted from an aqueous acidic solution into toluene via ion-pair formation with the protonated RBH(+) ion. Upon mixing the extract with a reversed micellar reagent solution of cetyltrimethylammonium chloride (CTAC) in 1-hexanol-cyclohexane/water (0.60 mol dm(-3) H(2)SO(4)) containing cerium(IV), uptake of the ion-pair by CTAC reversed micelles occurred easily, followed by an oxidation reaction of RB with Ce(IV) in the CL process. The CL signal produced was then measured. Using a flow injection system, the detection limits (DL) of 0.25 μmol dm(-3) Sb(III) and 0.20 μmol dm(-3) Sb(V), and linear calibration graphs with dynamic ranges from the respective DLs to 16 μmol dm(-3) for Sb(III) and Sb(V) were obtained under optimized experimental conditions. The proposed method was successfully applied to a mixture of Sb(III) and Sb(V), where total antimony, Sb(III) + Sb(V), was measured using ceric sulfate as an oxidant to oxidize Sb(III) to Sb(V) prior to extraction, Sb(V) was determined directly without the use of an oxidant, and Sb(III) was calculated by difference.

Сорбционно-хроматографическое определение галловой, кофейной кислот, рутина и эпикатехина в лекарственных растениях

The sorption processes of solid-phase extraction of phenolic compounds from water extracts of medicinal herbs followed by chromatographic technique have been studied. Using model aqueous solutions and water extracts of Hypericum perforatum and Achillea millefolium the Diapak C18 recoveries overcome versus Diapak P (hypercross-linked polystyrene) and liquid-liquid extraction technique. The conditions of sorption extraction and determination of gallic acid, trans-caffeic acid and rutin using the sorbent Diapak C18 were optimized. Key words : solid-phase extraction (SPE), high-performance liquid chromatography (HPLC), phenolic compounds, medicinal herbs (Russian) DOI: http://dx.doi.org/10.15826/analitika.2013.17.2.012 Z.A. Temerdashev, V.V. Milevskaya, N.V. Kiseleva, N.A. Vernikovskaya, V. А . Korobkov Kuban State University, Krasnodar, Russian Federation

UHPLC/ESI‐Q‐TOF‐MS method for the measurement of dopamine in rodent striatal tissue: A comparative effects of intranasal administration of ropinirole solution over nanoemulsion

An ultra high performance liquid chromatography-electrospray ionization-tandem mass spectrometric method (UHPLC/ESI-Q-TOF-MS) for the analysis of dopamine (DA) in Wistar rat brain homogenate has been developed and validated. The chromatographic separation was achieved on a Waters ACQUITY UPLC™ BEH C18 (100.0 mm × 2.1 mm; 1.7 µm) column using isocratic mobile phase, consisting of acetonitrile and Formic acid (0.1% w/v) (10: 90; v/v), at a flow rate of 0.15 ml min(-1) . The transitions occurred at m/z 154.04 → 137.006 for DA, and m/z 184.204 → 166.08 for the internal standard. The recovery of the analytes from Wistar rat brain homogenate was optimized using liquid-liquid extraction technique (LLE) in ethyl acetate. The total run time was 3.5 min and the elution of DA occurred at 1.44 ± 0.05 min. The linear dynamic range was established over the concentration range 75-750 ng mL(-1) (r(2) ; 0.9921 ± 0.0005) for DA. The intra-assay and inter-assay accuracy in terms of % CV was in the range 0.73-2.80. The lower limit of detection (LOD) and quantitation (LOQ) for DA was 0.278 and 0.844 ng mL(-1) , respectively. Analytes were stable under various conditions (in autosampler, during freeze-thaw, at room temperature, and under deep-freeze conditions). The developed method was successfully applied for in vivo profiling in rodents.

Distribution of U(VI) from Aqueous Solutions into Chloroform Solution of N,N’-Ethylenebis (4-butanoyl-2,4-dihydro-5-methyl-2-phenyl-3H-pyrazol-3-oneimine) Schiff Base

The distribution of U(VI) from aqueous media into chloroform solution of N,N’-ethylenebis (4-butanoyl-2,4-dihydro-5-methyl-2-phenyl-3H-pyrazol-3-oneimine) (H2BuEtP) a Schiff base has been studied to determine the effect of buffer media, mineral acids, auxiliary complexing agents and 4-butanoyl-2,4-dihydro-5-methyl-2-phenyl-3H-pyrazol-3-one (HBuP) on the distribution behaviour. A 1:1 interaction was observed between U(VI) and the ligand during the extraction process and one mole of hydrogen ion was found to have been displaced in the reaction process. The study shows that pH½ of the extraction of U(VI) shifted from 4.88 ± 0.10 to 2.14 ± 0.10 for CHCl3 solutions of H2BuEtP and H2BuEtP-HBuP respectively. Extraction constants (Log Kex) -1.68 ± 0.12 and -0.72 ± 0.13 were determined respectively from the studies, indicating that addition of HBuP made the extraction process more efficient. Species extracted were characterized as complexes with suggested formulae UO2(HBuEtP)X and UO2(HBuEtP)X.HBuP where X is an anion. Graphical analysis shows also that UO2(HBuEtP)X.HBuP could be extracted as UO2(BuP)X.(H2BuEtP), indicating a potential synthetic method for metal complex species with position isomers by liquid-liquid extraction technique. Studies of extraction from auxiliary complexing agents support the existence of species proposed above leading to enhanced extraction of U(VI) from X = NO3-, SO42, CH3COO-,SCN-, Cl-, I- and F- media. Percentage extraction of U(VI) from mineral acids suggests that U(VI) extraction tend to increase with decrease in acid strength.

Bromelain purification through unconventional aqueous two-phase system (PEG/ammonium sulphate)

This paper focuses on the feasibility of unconventional aqueous two-phase systems for bromelain purification from pineapple processing waste. The main difference in comparison with conventional systems is the integration of the liquid-liquid extraction technique with fractional precipitation, which can decrease the protein content with no loss of biological activity by removing of unwanted molecules. The analysis of the results was based on the response surface methodology and revealed that the use of the desirability optimisation methodology (DOM) was necessary to achieve higher purification factor values and greater bromelain recovery. The use of DOM yielded an 11.80-fold purification factor and 66.38 % biological activity recovery using poly(ethylene glycol) (PEG) with a molar mass of 4,000, 10.86 % PEG concentration (m/m) and 36.21 % saturation of ammonium sulphate.

METHOD OF ANALYSIS VALIDATION OF DIOXIN-LIKE PCB’S IN DAIRY PRODUCT

Dioxin like-polychlorinated biphenyls are unintentionally produced ubiquitous, persistent organic pollutants. The main source of human exposure to the compounds is food of animal origin because bioaccumulation in food chains. Extraction of dairy products by liquid-liquid extraction (LLE) technique for milk but extraction of other dairy products such as cheese was by soxhlet technique. In addition, clean samples after extracting the fat from dairy product samples by three - column chromatographyies, which include multi-layer silica, alumina column and then the third phase of the chromatographic column, is carbon. Method of analysis for determination of “dioxin-like PCBs” -show high toxicological properties that are similar to dioxins - in dairy products by high-resolution gas chromatography/ high-resolution mass spectrometry (HRGC/HRMS). Results were development of reliable and validated analytical method for dairy products, which recognized to be good indicator of environmental exposure for persistent organic pollutants. The method performance was tested for four non-ortho PCB congeners (PCB 77, 81,126, and 169) and eight mono-ortho PCB congeners (105, 114, 118, 123, 156, 157, 167, and 189) with good average recovery (via 13C12-labelled PCB internal standard) of the twelve PCB congeners for dairy products at level 40 ng/kg varies between 99-109 %. The trueness of a measurement method for dioxin like polychlorinated biphenyls in dairy product such as cheese where the results demonstrated satisfaction z-scores within range of ±2 for sum non-ortho-PCB was -0.85, and for sum mono-ortho-PCB -0.74. The reproducibility expressed as relative standard deviation percent was less than 22.6 % and the measured uncertainty including random and systemic error (on 95% confidence level) was less than 40%.

High-Performance Liquid Chromatographic Assay for Ziprasidone in Plasma Samples: Application to Pharmacokinetic Studies in Rats

Ziprasidone (ZRS) is among the various antipsychotic drugs indicated for treating schizophrenia. The determination of the pharmacokinetic behavior of this drug is of utmost importance in evaluating its bioavailability. The objectives of the present study are: (1) to develop and validate a sensitive, specific, accurate and precise reverse-phase high performance liquid chromatographic method for quantification of ZRS in the plasma of rats; and (2) to apply the developed method to study the pharmacokinetic profile of ZRS in rats after oral administration. The method uses a C18 (250.0 × 4.6 mm, 5 µm) column and ultraviolet detector with wavelength set at 210.0 nm. The mobile phase is acetonitrile-phosphate buffer (pH 3.6) 28:72% v/v at a flow rate of 1.0 mL/min. The internal standard (IS) is escitalopram. The extraction procedure for ZRS and IS from the biological matrix (plasma) employs liquid-liquid extraction technique using a mixture of methyl tert-butyl ether-dichloromethane (70:30% v/v). The results show good accuracy and precision over a linearity range of 20.0-3,000.0 ng/mL with r(2) ≥ 0.9986. The mean recoveries of ZRS and IS are 79.32 ± 1.16 and 84.10 ± 3.2%, respectively. The method has been successfully utilized to study the pharmacokinetic profile of ZRS in rats after oral administration.

Development and Validation of UHPLC/ESI-Q-TOF-MS Method for Terbutaline Estimations in Experimental Rodents: Stability Effects and Plasma Pharmacokinetics

An ultra high performance liquid chromatography-electrospray ionization-tandem mass spectrometric method (UHPLC/ESI-Q-TOF-MS) for the analysis of terbutaline (TB) in Wistar rat plasma has been developed and validated. The chromatographic separation was achieved on a Waters ACQUITY UPLCTM BEH C18 (100.0 mmx2.1 mm; 1.7 μm) column using isocratic mobile phase, consisting of 2 mM ammonium acetate and acetonitrile (90: 10; v/v), at a flow rate of 0.25 mL min-1. The transitions occurred at m/z 226.19→152.12 for TB, and m/z 260.34→183.11 for the internal standard. The recovery of the analytes from Wistar rat plasma was optimized using liquid- liquid extraction technique (LLE) in ethyl acetate. The total run time was 3.0 min and the elution of TB occurred at 1.85±0.05 min. The linear dynamic range was established over the concentration range 1–1000 ng mL-1 (r2; 0.9938±0.0005) for TB. The intra-assay and inter-assay accuracy in terms of % CV was in between 1.8–3.5. The lower limit of quantitation (LLOQ) for TB was 1.0 ng mL-1. Analytes were stable under various conditions (in autosampler, during freeze–thaw, at room temperature, and under deepfreeze conditions). The developed method was successfully applied for pharmacokinetic profiling in rodents. Keywords: Asthma, Bronchodilator, Ex-vivo Stability, In-vitro assay, Matrix effect, Pharmacokinetics, Terbutaline; UHPLC/ESI-Q-TOF-MS, Validation

Simultaneous determination of sitagliptin and simvastatin in human plasma by LC‐MS/MS and its application to a human pharmacokinetic study

A simple, rapid and sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) assay method has been developed and validated for simultaneous quantification of sitagliptin and simvastatin in human plasma. Carbamazepine was used as an internal standard (IS). The analytes and IS were extracted from the human plasma by liquid-liquid extraction technique. The reconstituted samples were chromatographed on an Alltima HP C(18) column using an isocratic solvent mixture [acetonitrile-5 mm ammonium acetate (pH 4.5), 85:15 (v/v)] at a flow rate of 1.0 mL/min. Method validation was performed as per Food and Drug Administration guidelines and the results met the acceptance criteria. The calibration curves obtained were linear (r(2) ≥ 0.99) over the concentration range of 0.10-501 and 0.05-105 ng/mL for sitagliptin and simvastatin, respectively. The results of the intra- and inter-day precision and accuracy studies were well within the acceptable limits. Both the analytes were found to be stable in a battery of stability studies. The method is precise and sensitive enough for its intended purpose. A run time of 3.0 min for each sample made it possible to analyze more than 300 plasma samples per day. The developed assay was successfully applied to a pharmacokinetic study in human volunteers.

Determination of febuxostat in human plasma using ultra‐performance liquid chromatography tandem mass spectrometry

A rapid and sensitive liquid chromatography-tandem mass spectrometry method has been developed and validated for the determination of febuxostat in human plasma. The liquid-liquid extraction technique was used for the extraction of febuxostat from human plasma using trandolapril as the internal standard (IS). Chromatography was performed on a ultra-performance liquid chromatography (UPLC) BEH C18, 50 mm X 2.1 mm, 1.7 µm particle size column, with the mobile phase consisting of 0.1% formic acid and acetonitrile (in a 25:75 ratio), followed by detection using mass spectrometry. The method involves a simple reversed isocratic chromatography condition and mass spectrometry detection, which enables detection at sub-microgram levels. The method was validated and the lower limit of quantification for febuxostat was found to be 0.075 µg/ml. The mean recovery for febuxostat ranged from 100.9 to 106.5%. This method increased the sensitivity and selectivity; resulting in high-throughput analysis of febuxostat using commercially available IS for pharmacokinetic, bioavailability, and bioequivalence studies, with a chromatographic run time of 1.5 min only.

A rapid and sensitive liquid chromatography–tandem mass spectrometric assay for moexipril, an angiotensin‐converting enzyme inhibitor in human plasma

A simple, rapid and sensitive liquid chromatography/tandem mass spectrometry method was developed and validated for the quantification of angiotensin-converting enzyme inhibitor, moexipril, in human plasma. Benazepril was used as an internal standard (IS). Analyte and IS were extracted from the human plasma by liquid-liquid extraction technique using ethyl acetate. The reconstituted samples were chromatographed on a C₁₈ column by using a mixture of methanol and 0.1% formic acid buffer (85:15, v/v) as the mobile phase at a flow rate of 0.5 mL/min. The calibration curve obtained was linear (r ≥ 0.99) over the concentration range of 0.2-204 ng/mL. The multiple reaction-monitoring mode was used for quantification of ion transitions at m/z 499.4/234.2 and 425.2/351.1 for moexipril and IS, respectively. The results of the intra- and inter-day precision and accuracy studies were well within the acceptable limits. A run time of 2.0 min for each sample made it possible to analyze more than 400 plasma samples per day. The proposed method was found to be applicable to clinical studies.