Simultaneous analysis of bioactive metabolites from Ziziphus jujuba by HPLC–DAD–ELSD–MS/MS

A high-performance liquid chromatography (HPLC) with diode array detector (DAD), evaporative light scattering detector (ELSD) and electrospray ionization mass spectrometry (ESI-MS) was established for the simultaneous determination of nine representative metabolites of the alkaloid, flavonoid and triterpenoid classes from Ziziphus jujuba var. spinosa. The optimal chromatographic conditions were obtained on an ODS column and the mobile phase was composed of water and methanol with 0.1% formic acid using a gradient elution system. Using the developed methods, all of the validation parameters were successfully obtained. In addition, effectiveness of diverse extraction methods was compared to each other for the development of standard analytic method. The verified method was successfully applied to the quantitative determination of representative metabolites in commercial samples of Z. jujuba and Z. mauritiana from different markets in Korea, China and Myanmar. The analytical results showed that the contents of the nine analytes vary significantly with sources and species, thus demonstrating its potential for the detection of this plant.     

Determination of mangiferin in rat plasma by liquid–liquid extraction with UPLC–MS/MS

An ultra-performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS) method employing electrospray ionization (ESI) has been developed for the determination of mangiferin in rat plasma using diphenhydramine as the internal standard (IS). Liquid–liquid extraction (LLE) was used for sample preparation and the analysis was achieved with gradient elution on C₁₈ reversed phase column. The method was validated over the concentration range 0.02–5.0 μg/mL for oral administration and 0.4–100 μg/mL for intravenous administration. The intra-day and inter-day precision of mangiferin expressed as RSD < 15% and the accuracy (RE) did not exceed 15%. This validated method is a novel technique for sample preparation and quantitation, which was successfully applied to estimate the bioavailability of mangiferin.     

LC–MS/MS studies of ritonavir and its forced degradation products

Forced degradation of ritonavir (RTV), under the conditions of hydrolysis (acidic, basic and neutral), oxidation, photolysis and thermal stress as prescribed by ICH was studied using LC–MS/MS. Eight degradation products were formed and their separation was accomplished on Waters XTerra^® C₁₈ column (250 mm × 4.6 mm i.d., 5 μm) using water:methanol:acetonitrile as (40:20:40, v/v/v) mobile phase in an isocratic elution mode by LC. The method was extended to LC–MS/MS for characterization of the degradation products and the pathways of decomposition were proposed. No previous reports were found in the literature regarding the characterization of degradation products of ritonavir.     

Identification of multiple constituents in the traditional Chinese medicine formula GuiZhiFuLing-Wan by HPLC–DAD–MS/MS

GuiZhiFuLing-Wan (GFW) has been used in China for centuries to improve blood stagnation. In this paper, a HPLC–DAD–MS/MS method was established for the efficient and rapid identification of the chemical constituents in extract of GuiZhiFuLing-Wan. Separation was performed on an Alltima C₁₈ analytical column by gradient elution with CH₃CN/H₂O–CH₃COOH as mobile phase at a flow rate 1.0 ml/min. 27 potentially bioactive compounds including monoterpene glycosides, galloyl glucoses, acetophenones, phenylallyl compounds and triterpenoids were identified or tentatively characterized by online ESI/MS/MS and the comparison with literature data and authentic compounds. After the identification, six different brands of GFW commercial products in various dosage forms were evaluated. The results demonstrated that capsule of GFW was superior to the other two dosage forms, honeyed pill and concentrated pill in administration. The points that should be paid more attention during the manufacturing process of GFW were also analyzed. The method can be the basis for the quality control of this commonly used herbal formula.     

Development of a simultaneous LC–MS/MS method to predict in vivo drug–drug interaction in mice

Cocktail substrates are useful in investigating drug–drug interactions (DDI) that can rapidly identify the cytochrome P450 (CYP) isoforms that interact with test drugs. In this study, we developed and validated five probe drugs for CYP1A, CYP2B, CYP2C, CYP2D, and CYP3A using LC–MS/MS to determine CYP activities in mice. The five probe substrates were caffeine (2 mg/kg), bupropion (30 mg/kg), omeprazole (4 mg/kg), dextromethorphan (40 mg/kg), and midazolam (2 mg/kg) for CYP1A, CYP2B, CYP2C, CYP2D, and CYP3A, respectively. The cocktail substrates were orally administered to male 5-week-old ICR mice over 0–240 min. The analytical method was validated; it showed high selectivity, linearity, and acceptable accuracy. We confirmed the lack of interaction of this cocktail in the control state (no effect of CYP inducer or inhibitor) and suggested AUC_ratio (metabolite/substrate) as a unit to evaluate DDI in vivo. In addition, the cocktail assay was applied for the determination of pharmacokinetic parameters against phenobarbital as a selective CYP2B inducer and ketoconazole as a strong CYP3A inhibitor. The concentration of cocktail substrates and the LC–MS/MS method were optimized. In conclusion, we developed a simultaneous and comprehensive analysis system for predicting potential DDI in mice.     

Determination of fluoroquinolones in chicken feces – A new liquid–liquid extraction method combined with LC–MS/MS

The application of antibiotics including fluoroquinolones to farming animals is widespread and may lead to the development of antibiotic resistance and other environmental effects. To calculate environmental loads and for a proper risk assessment it is necessary to determine the antibiotic concentration in feces. Therefore, a new liquid–liquid extraction method combined with HPLC–MS/MS for the detection of marbofloxacin, ciprofloxacin, enrofloxacin and difloxacin in chicken feces was developed. Recoveries ranged from 51.0% to 83.5%. LOQs were between 0.10 and 1.09 μg/kg. Feces of chickens treated with an enrofloxacin dosage of 10 mg/kg bodyweight revealed maximum enrofloxacin and ciprofloxacin concentrations of 61.3 and 18.8 mg/kg. Both antibiotics could be detected in feces up to two days after the last application in notable amounts (∼1 mg/kg). Thus, feces of recently medicated chickens should not be used as a fertilizer without any further processing.     

Direct quantitation of glucoraphanin in dog and rat plasma by LC–MS/MS

A rapid method to quantify levels of the β-thioglycoside N-hydroxyl sulfate, glucoraphanin, in dog and rat plasma to support pre-clinical toxicological and pharmacological studies has been developed using liquid chromatography–tandem mass spectrometry (LC–MS/MS). Glucoraphanin was extracted from plasma by protein precipitation with acetonitrile and separated via hydrophilic interaction liquid chromatography (HILIC) using a Luna 5 μm Silica (2) 100 Å column (50 mm× 2.0 mm) at a flow rate of 0.3 mL/min. Solvent A consisted of 200 mM ammonium acetate and formic acid (99:1, v/v) and Solvent B was acetonitrile. Initial conditions (90% Solvent B) were held for 0.01 min after injection, decreased to 40% in 0.5 min and held constant for 2.5 min, returning to initial conditions for 3 min (reequilibration time). Glucoraphanin was detected by MS/MS using a turbo ion spray interface as the ion source operating in negative ion mode. Acquisition was performed in multiple reaction monitoring mode at m/z 435.8 → 96.7. The method was validated for the calibration range 10–2000 ng/mL. Within- and between-run precision for the low, mid and high QC levels was 8% R.S.D. or less and accuracy ranged from 100 to 113%. The lower limit of quantification was 10 ng/mL; calibration curves encompassed the range of plasma concentrations expected to be found in bioavailability and pharmacokinetics studies with glucoraphanin. The method has successfully been applied to the determination of glucoraphanin in dog and rat plasma and should be extendable to other species as well.     

HPLC–MS and HPLC–MS/MS analysis of seven active constituents of Xiao-Xu-Ming decoction and application to a pharmacokinetic study after oral administration to rat

Xiao-xu-ming decoction (XXMD) is a traditional Chinese medicine that has been widely used to treat theoplegia and its sequelae. This paper reports the development of three separate assays based on reversed phase high-performance liquid chromatography–mass spectrometry (HPLC–MS) and HPLC–MS/MS for the determination of seven active constituents of XXMD viz oroxylin A-7-O-glucuronide, wogonoside, liquiritigenin, cimifugin, 5-O-methylvisammiol, glycyrrhizic acid and glycyrrhetinic acid in rat plasma. All calibration curves were linear (r >0.99) with lower limits of quantitation (LLOQs)<12.4 ng/mL. Intra- and inter-day precisions (as relative standard deviation) were all <10.7% with recoveries in the range of 88.7–113%. In addition, the seven analytes were shown to be stable in rat plasma samples under relevant storage conditions. The validated methods were successfully applied to a pharmacokinetic study in rat after oral administration of XXMD.     

Separation and characterization of forced degradation products of abacavir sulphate by LC–MS/MS

Abacavir sulphate was subjected to forced degradation under the conditions of hydrolysis (acid, alkali and neutral), oxidation, photolysis and thermal stress as prescribed by ICH. Eight degradation products were formed and their separation was accomplished on Waters XTerra C₁₈ (250 mm × 4.6 mm, 5 μm) column using 20 mM ammonium acetate:acetonitrile as a mobile phase in gradient elution mode by LC. The degradation products were characterized by LC–MS/MS and its fragmentation pathways were proposed. No previous reports were found in the literature regarding the degradation behavior of abacavir sulphate.     

LC–MS/MS determination of carbamathione in microdialysis samples from rat brain and plasma

A selective liquid chromatography–tandem mass spectrometric (LC–MS/MS) method was developed for the determination of S-(N, N-diethylcarbamoyl) glutathione (carbamathione) in microdialysis samples from rat brain and plasma. S-(N, N-Diethylcarbamoyl) glutathione (carbamathione) is a metabolite of disulfiram. This metabolite may be responsible for disulfiram's effectiveness in the treatment of cocaine dependence. Chromatographic separations were carried out on an Alltech Altima C-18 (50 mm long × 2.1 mm i.d., 3 μm particles) analytical column at a flow rate of 0.3 ml/min. Solvent A consisted of 10 mM ammonium formate, methanol, and formic acid (99:1:0.06, v/v/v). Solvent B consisted of methanol, 10 mM ammonium formate and formic acid (99:1:0.06, v/v/v). A 20 min linear gradient from 95% aqueous to 95% organic was used. Tandem mass spectra were acquired on a Micromass Quattro Ultima “triple” quadrupole mass spectrometer equipped with an ESI interface. Quantitative mass spectrometric analysis was conducted in positive ion mode selected reaction monitoring (SRM) mode looking at the transition of m/z 407–100 and 175 for carbamathione and m/z 392–263 for the internal standard S-hexyl glutathione. The simultaneous collection of microdialysate from blood and brain was used to monitor carbamathione concentrations centrally and peripherally. Good linearity was obtained over a concentration range of 0.25–10,000 nM. The lowest limit of quantification (LLOQ) was determined to be 1 nM and the lowest limit of detection (LLOD) was calculated to be 0.25 nM. Intra- and inter-day accuracy and precision were determined and for all the samples evaluated, the variability was less that 10% (R.S.D.).     

HPLC–DAD–MS/MS profiling of standardized rosemary extract and enhancement of its anti-wrinkle activity by encapsulation in elastic nanovesicles

The anti-wrinkle activity of defatted rosemary extract (DER) was assessed, and its effect was optimized by encapsulation in transferosomes (TFs). DER was standardized to a rosmarinic acid content of 4.58 ± 0.023 mg% using reversed-phase high performance liquid chromatography (Rp-HPLC), and its components were identified by HPLC-diode array detection-tandem mass spectrometry. In vitro free radical scavenging assays showed DER had high free radical scavenging activity against 2,2-diphenyl-2-picryl hydrazyl, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), and superoxide radicals. DER also inhibited bleaching of β-carotene with high Fe(III) and Fe(II) chelating ability. In vivo anti-wrinkle activities of topically applied DER (20, 50, and 100 mg) and a TF formulation (TF4, 20 mg of DER) were evaluated in UVB-irradiated mice using a wrinkle scoring method, metalloproteinase (MMP) expression, and histopathology. Among the nanovesicles, TF4 was the most deformable, and had an acceptable size and encapsulation efficiency and enhanced permeation of DER through rat skin compared with unencapsulated DER. DER (50 and 100 mg) and TF4 significantly inhibited MMP-2 and MMP-9 expression and improved wrinkle scores. DER and TF4 moderately decreased epidermal thickness without pigmentation. DER is a potent natural antioxidant for combating skin aging. Moreover, encapsulation of DER in TFs will enhance its skin permeation and anti-wrinkle activity.     

HPLC–MS/MS测定赖诺普利血药浓度及其在药动学研究中的应用

目的 建立HPLC-MS/MS测定人血浆中赖诺普利的浓度并研究其药动学特征,为该药的临床应用提供依据。方法 20名健康受试者单剂量口服赖诺普利片20 mg后,采用HPLC-MS/MS测定其血药浓度,利用DAS软件对血药浓度-时间数据进行药动学模型拟合和参数计算,应用AIC法判别房室模型。结果 血浆中赖诺普利在2.0~200 ng·mL^-1内线性关系良好(r=0.997 5),平均回收率为88.8%,日内RSD≤6.62%,日间RSD≤13.0%。最佳房室模型为单室模型(Wi=1/C2, AIC=4.61),主要药动学参数t_1/2为(10.91±3.71)h,t_max为(6.65±1.50)h,C_max为(98.15±23.66)ng·mL^-1,Ka为(0.83±1.28)h^-1,Vd/F为(220.70±62.82)L,AUC_0-72为(1 437.41±399.68)ng·h·mL^-1,AUC_0-∞为(1 516.54±376.83)ng·h·mL^-1。结论 该分析方法专属性强、灵敏度高,适合大样本分析,满足临床血药浓度监测的要求并适用于药动学领域的研究。     

Development and validation of an HPLC–MS/MS method to determine clopidogrel in human plasma

A quantitative method for clopidogrel using online-SPE tandem LC–MS/MS was developed and fully validated according to the well-established FDA guidelines. The method achieves adequate sensitivity for pharmacokinetic studies, with lower limit of quantifications (LLOQs) as low as 10 pg/mL. Chromatographic separations were performed on reversed phase columns Kromasil Eternity-2.5-C18-UHPLC for both methods. Positive electrospray ionization in multiple reaction monitoring (MRM) mode was employed for signal detection and a deuterated analogue (clopidogrel-d₄) was used as internal standard (IS). Adjustments in sample preparation, including introduction of an online-SPE system proved to be the most effective method to solve the analyte back-conversion in clinical samples. Pooled clinical samples (two levels) were prepared and successfully used as real-sample quality control (QC) in the validation of back-conversion testing under different conditions. The result showed that the real samples were stable in room temperature for 24 h. Linearity, precision, extraction recovery, matrix effect on spiked QC samples and stability tests on both spiked QCs and real sample QCs stored in different conditions met the acceptance criteria. This online-SPE method was successfully applied to a bioequivalence study of 75 mg single dose clopidogrel tablets in 48 healthy male subjects.Abbreviations: ESI, electrospray ionization; IS, internal standard; LC–MS/MS, liquid chromatography tandem mass spectrometry; LLOQ, lower limit of quantification; MRM, multiple reaction monitoring; SPE, solid phase extraction; QC, quality controlKEY WORDS: Clopidogrel, Online-SPE, LC–MS/MS, Back-conversion, Bioequivalence, Deuterated analogue, Real samples stability, FDA guidelines     

Determination of tramadol and metabolites by HPLC-FL and HPLC–MS/MS in urine of dogs

Tramadol is a centrally acting analgesic drug used in veterinary and human clinical practice. Its metabolism has been largely characterized in human being but is still long to be comprehended in several animal species, especially in the dog. The aim of the present study was to develop and validate a new analytical procedure to investigate HPLC the metabolization/elimination process tramadol in urine of dogs by HPLC-FL or HPLC–MS/MS. A single oral dose of tramadol (4 mg/kg) was administered to 4 male Beagle dogs and the urine was naturally collected. This matrix either hydrolyzed than un-hydrolyzed was extracted with different blends of solvents to detect the total or free form of the analytes, respectively.     

A novel LC–MS/MS assay for methylprednisolone in human plasma and its pharmacokinetic application

Methylprednisolone is a synthetic glucocorticoid. In our report, the authors proposed a sensitive and selective liquid chromatography/tandem mass spectrometry (LC–MS/MS) assay for the determination of methylprednisolone applying budesonide as internal standard. Liquid–liquid extraction (LLE) having tert–butyl methyl ether (TBME) have been employed to extract methylprednisolone from the plasma samples. Immediately after reconstitution, the samples were chromatographed on a C₁₈ column using an isocratic mobile phase composed of 10 mM ammonium formate buffer and acetonitrile (35:65, v/v). A flow rate of 1.00 ml/min was used to elute the analyte form the column. Analysis was carried out with an API–4000 LC–MS/MS instrument operated in multiple reaction-monitoring (MRM) mode. The linearity has been proven within the concentration range of 10.1–804 ng/ml in plasma samples. The precision (%CV) and accuracy results in five validation batches across five concentration levels were well within the acceptance limits. The drug was stable under different conditions. The particular assay has been proficiently put on pharmacokinetic study in healthy male subjects.     

Analysis of bioactive compounds using LC–ESI–MS/MS,cytotoxic, antimicrobial effects,and enzyme activities from Cyclotrichium origanifolium

Cyclotrichium origanifolium is a medicinal plant belonging to the Lamiaceae family. In this study, phenolic content analysis, antimicrobial effects, and cytotoxic effects of extracts of C. origanifolium were investigated. In the extracts, phenolic compound analysis by the liquid chromatography–electrospray ionization–tandem mass spectrometry method, antimicrobial effect by the minimum inhibition concentration method, and cytotoxic effect on human dermal fibroblasts (HDF), glioblastoma cell (U87), ovarian adenocarcinoma cell (Skov-3), and human colorectal adenocarcinoma cell (CaCo-2) cancer cell lines were investigated. Cytotoxicity analyses were performed by the MTT method. In addition, the GST and AChE enzyme activities of the extracts were also measured. Around 18 compounds were detected in both the methanol and ethanol extract. It was found that the best antimicrobial effect on Gram-negative Pseudomonas aeruginosa was on methanol extract, while the ethanol extract was on Candida albicans fungus (respectively, 2.50 mg/ml, 5.0 μg/ml). A 500 μg/ml of methanol extract has been shown to have cytotoxic activity high effect on HDF cells. GST and AChE activity were found to decrease in a concentration-dependent manner.     

Determination of rat plasma levels of sertraline enantiomers using direct injection with achiral–chiral column switching by LC–ESI/MS/MS

A highly sensitive and selective on-line two-dimensional reversed-phase liquid chromatography/electrospray ionization–tandem mass spectrometric (2D-LC–ESI/MS/MS) method to determine sertraline (SRT) enantiomers in rat plasma was developed and validated. The method was applied to separate and determine the diastereomers and enantiomers of SRT simultaneously. The 2D-LC–ESI/MS/MS system consisted of RAM column in first dimension for trapping proteineous part of plasma and a chiral Cyclobond column as second dimension for separation of enantiomers and diastereomers of SRT using 0.1% aqueous trifluoroacetic acid:acetonitrile (86:14, v/v) as mobile phase in an isocratic elution mode. The linear dynamic range was 0.5–200 ng/mL (r² > 0.999). Acceptable precision and accuracy were obtained over the calibration range. The assay was successfully used in the analysis of SRT enantiomers in rat plasma to support pharmacokinetic studies.     

Quality assessment of traditional herbal formula,Hyeonggaeyeongyo-tang through simultaneous determination of twenty marker components by HPLC–PDA and LC–MS/MS

Simultaneous analysis of 20 marker components (gallic acid, cimifugin, geniposide, paeoniflorin, ferulic acid, nodakenin, narirutin, naringin, neohesperidin, arctiin, baicalin, oxypeucedanin hydrate, wogonoside, baicalein, arctigenin, glycyrrhizin, wogonin, pulegone, decursin, and decursinol angelate) for quality assessment of the traditional herbal formula, Hyeonggaeyeongyo-tang (HYT) was carried out by using high-performance liquid chromatography (HPLC) with photodiode array detection (PDA) and liquid chromatography–mass spectrometry with tandem mass spectrometry (LC–MS/MS). The coefficient of determination showed excellent linearity of more than 0.9999 for all analytes. The recovery of 20 marker components was 93.92 to 102.66% with relative standard deviation (RSD) < 3.00% and RSD value of precision was ≤ 3.44%. The amounts of 20 marker components using HPLC–PDA and LC–MS/MS were determined to be 0.18–14.60 and 0.01–1.76 mg/freeze-dried g, respectively.     

LC–MS/MS assay for olanzapine in human plasma and its application to a bioequivalence study

This paper describes a selective and sensitive assay for the determination of olanzapine (OLZ) in human plasma based on liquid chromatography–tandem mass spectrometry (LC–MS/MS). The analyte and quetiapine as internal standard (IS) were extracted from 200 μL plasma via solid phase extraction on Waters Oasis HLB cartridges. Chromatographic separation was achieved on an ACE 5C18-300 column (100 mm×4.6 mm, 5 μm) under isocratic conditions in a run time of 3.5 min. Mass spectrometric detection involved electrospray ionization in the positive ion mode followed by multiple reaction monitoring (MRM) of the transitions at m/z 313/256 for OLZ and m/z 384/253 for the IS. The assay was linear in the range 0.10–40.0 ng/mL with a lower limit of quantitation and limit of detection of 0.10 and 0.012 ng/mL, respectively. Intra- and inter-day precision (as coefficient of variation) and relative recovery were <5.0% and >90%, respectively. The method was successfully applied to a bioequivalence study of 5 and 10 mg OLZ disintegrating tablets in 40 healthy Indian males with reproducibility by incurred sample reanalysis in the range ?7.43 to 8.07%.     

Identification and characterization of degradation products of irbesartan using LC–MS/TOF,MS, on-line H/D exchange and LC–NMR

Irbesartan was subjected to hydrolytic, oxidative, photolytic and thermal stress, according to ICH guideline Q1A (R2). The drug showed degradation only in acidic, basic and photoacidic conditions, while it was stable to other stress conditions. A total of three degradation products were formed, which were separated on a C-8 column employing a gradient HPLC method. Initially, a complete mass fragmentation pathway of the drug was established with the help of MS/TOF, MSⁿ and H/D exchange studies. Subsequently, the degradation products were subjected to LC–MS/TOF and on-line H/D exchange mass studies to obtain their accurate mass, fragment pattern and number of labile hydrogens. The MS results helped to assign tentative structures to degradation products, which were verified through ¹H and 2D COSY LC–NMR experiments. The products were identified as (2′-(1H-tetrazol-5-yl)biphenyl-4-yl)methanamine, 1-(1-((2′-(1H-tetrazol-5-yl)biphenyl-4-yl)methylamino)pentylideneamino)cyclopentane carboxylic acid and 2-butyl-3-(tetrazolo[1,5-f]phenanthridin-6-ylmethyl)-1,3-diazaspiro[4.4]non-1-en-4-one. The structures were justified by mechanisms of their formation.