Plasma lipid analysis by hydrophilic interaction liquid chromatography coupled with electrospray ionization tandem mass spectrometry

A novel method for the analysis of endogenous lipids and related compounds was developed employing hydrophilic interaction liquid chromatography with electrospray ionization tandem mass spectrometry. A hydrophilic interaction liquid chromatography with carbamoyl stationary phase achieved clear separation of phosphatidylcholine, lysophosphatidylcholine, sphingomyelin, ceramide, and mono‐hexsosyl ceramide groups with good peak area repeatability (RSD% < 10) and linearity (R² > 0.99). The established method was applied to human plasma assays and a total of 117 endogenous lipids were successfully detected and reproducibly identified. In addition, we investigated the simultaneous detection of small polar metabolites such as amino and organic acids co‐existing in the same biological samples processed in a single analytical run with lipids. Our results show that hydrophilic interaction liquid chromatography is a useful tool for human plasma lipidome analysis and offers more comprehensive metabolome coverage.     

Determination of clindamycin in animal plasma by high-performance liquid chromatography combined with electrospray ionization mass spectrometry

A method for the quantification of clindamycin in animal plasma using high-performance liquid chromatography combined with electrospray ionization mass spectrometry (LC/ESI-MS/MS) is presented. Lincomycin is used as the internal standard. The sample preparation includes a simple deproteinization step with trichloroacetic acid. Chromatographic separation is achieved on an RP-18 Hypersil column using gradient elution with 0.01 M ammonium acetate and acetonitrile as mobile phase. Good linearity was observed in the range 0-10 microg ml(-1). The limit of quantification of the method is 50 ng ml(-1) and the limit of detection is 1.3 ng ml(-1). The method was shown out to be of use for pharmacokinetic studies of clindamycin formulations in dogs.     

Simultaneous quantification of oleuropein and its metabolites in rat plasma by liquid chromatography electrospray ionization tandem mass spectrometry

Oleuropein (OE) is the cardinal bioactive compound derived from Olea europaea and possesses numerous beneficial properties for human health. However, despite the plethora of analytical methods that have studied the biological fate of olive oil‐derived bioactive compounds, no validated methodology has been published to date for the simultaneous determination of OE, along with all its major metabolites. In this study, a liquid chromatography‐electrospray ionization‐tandem mass spectrometry (LC‐ESI MS/MS) method has been developed and validated for the quantification of OE, simultaneously with its main metabolites hydroxytyrosol, 2‐(3,4‐dihydroxyphenyl)acetic acid, 4‐(2‐hydroxyethyl)‐2‐methoxy‐phenol or homovanillyl alcohol, 2‐(4‐hydroxy‐3‐methoxyphenyl)acetic acid or homovanillic acid, and elenolic acid in rat plasma matrix. Samples were analyzed by LC‐ESI MS/MS prior to and after enzymatic treatment. A solid‐phase extraction step with high mean recovery for all compounds was performed as sample pretreatment. Calibration curves were linear for all bioactive compounds over the range studied, while the method exhibited good accuracy, intra‐ and inter‐day precision. The limit of detection was in the picogram range (per milliliterof plasma) for HT and OE and in the nanogram range (per milliliter of plasma) for the other analytes, and the method was simple and rapid. The developed methodology was successfully applied for the simultaneous quantification of OE and its aforementioned metabolites in rat plasma samples, thus demonstrating its suitability for pharmacokinetics, as well as bioavailability and metabolism studies. Copyright © 2009 John Wiley & Sons, Ltd.     

Profiling histidine-containing dipeptides in rat tissues by liquid chromatography/electrospray ionization tandem mass spectrometry

The histidine-containing dipeptides carnosine (CAR) and structurally related anserine (ANS) and homocarnosine (HCAR), widely distributed in vertebrate organisms, have recently been proposed as endogenous quenchers for highly cytotoxic alpha,beta-unsaturated aldehydes generated by peroxidation. A sensitive, selective, specific and rapid liquid chromatographic/electrospray ionization tandem mass spectrometric assay was developed and validated for the simultaneous determination of these peptides in biological matrices in order to establish their plasma/tissue distribution. Samples (plasma or tissue homogenates from male rats) were prepared by protein precipitation with HClO(4) (1 : 1, v/v) containing H-Tyr-His-OH as internal standard. The supernatant was separated on a Phenomenex Sinergy polar-RP column with a mobile phase of water-acetonitrile-heptafluorobutyric acid (9 : 1 : 0.01, v/v/v) at a flow-rate of 0.2 ml min(-1), with a run time of 10 min. Detection was effected on an ion trap mass spectrometer equipped with an electrospray ionization interface operating in positive ionization mode. The acquisitions were in the multiple reaction monitoring mode using the following precursor --> product ion combinations: H-Tyr-His-OH (internal standard) m/z 319 --> 301; CAR m/z 227 --> 210 + 209; ANS m/z 241 --> 224 + 197 + 170; HCAR m/z 241 --> 156. The method was validated over the concentration range 15-1000 nmol g(-1) and the limit of quantification (LOQ) and limit of detection (LOD) were 12.5 and 4.2 pmol injected, respectively. The intra- and inter-day precisions were <10% (< or =17.47% at the LOQ) and the intra- and inter-assay accuracies were within +/-10% for all concentrations. The mapping profile in rat tissue gave the following results: the highest concentrations of CAR and ANS were found in skeletal muscles (soleus, gastrocnemius, tibialis), followed by the heart, cerebellum and brain (ANS below the LOQ). HCAR was found only in the brain and cerebellum. No histidine-containing dipeptides were detectable in plasma, liver, kidney and lung.     

Quantitative determination of domperidone in human plasma by ultraperformance liquid chromatography with electrospray ionization tandem mass spectrometry

A simple and sensitive liquid chromatography-tandem mass spectrometry method was developed and validated for determining domperidone in human plasma. The analyte and internal standard (IS; mosapride) were isolated from plasma samples by protein precipitation with methanol (containing 0.1% formic acid). The chromatographic separation was performed on an Xterra MS C(18) Column (2.1 x 150 mm, 5.0 microm) with a gradient programme mobile phase consisting of 0.1% formic acid and acetonitrile at a flow rate of 0.30 mL/min. The total run time was 4.0 min. The analyses were carried out by multiple reaction monitoring using the parent-to-daughter combinations m/z 426 --> 175 and m/z 422 --> 198 (IS). The areas of peaks from the analyte and IS were used for quantification of domperidone. The method was validated according to the FDA guidelines on bioanalytical method validation. Validation results indicated that the lower limit of quantification was 0.2 ng/mL, and the assay exhibited a linear range of 0.2-60.0 ng/mL and gave a correlation coefficient (r(2)) of 0.999 or better. Quality control samples (0.4, 0.8, 15 and 50 ng/mL) in six replicates from three different analytical runs demonstrated an intra-assay precision (RSD) 4.43-6.26%, an inter-assay precision 5.25-7.45% and an overall accuracy (relative error) of <6.92%. The method can be applied to pharmacokinetic and bioequivalence studies of domperidone.     

Analysis of saikosaponins in rat plasma by anionic adducts-based liquid chromatography tandem mass spectrometry method

Saikosaponins (SSs) are a class of triterpene saponins with a wide spectrum of bioactivities. A sensitive liquid chromatography tandem mass spectrometry (LC‐MS/MS) method was developed for simultaneous determination of saikosaponin a, saikosaponin c, saikosaponin d and saikosaponin b₂ in rat plasma. Plasma samples were prepared by liquid–liquid extraction. The analytes and the internal standard (IS) digoxin were well separated on an octadecyl column using gradient elution and analyzed by monitoring the fragmentation transition pair of anionic adducts to deprotonated molecules in negative‐mode electrospray. By neutral loss of HCOOH, the transition pairs of m/z 825 → 779 for SSa, SSd, SSb₂ and the IS, and m/z 971 → 925 for SSc were sensitive for MS/MS detection with the lower limits of quantification in the range of 0.20–0.40 ng/mL. Method validation experiments were performed, including selectivity, precision, accuracy, linearity, matrix effect, recovery and stability. The validated method was further applied to determine the pharmacokinetics parameters of SSa, c and d in rats following a single oral administration of the extract of chaihu (the dried roots of Bupleurum chinense DC). Copyright © 2011 John Wiley & Sons, Ltd.     

Determination of teniposide in rat plasma by ultra performance liquid chromatography electrospray ionization tandem mass spectrometry after intravenous administration

A novel, specific and rapid ultra performance liquid chromatography electrospray ionization tandem mass spectrometry method has been developed and validated for determination of teniposide in rat plasma. A one‐step liquid–liquid extraction method was used and the separation was carried out on an Acquity UPLC^TM BEH C₁₈ column with gradient elution using a mobile phase composed of acetonitrile and water (containing 0.1% formic acid) at a flow rate of 0.20 mL/min. A triple quadrupole tandem mass spectrometer in multiple‐reaction monitoring mode via an electrospray ionization interface was used for the detection of teniposide. The detection was complete within 3.0 min. A linear calibration curve was obtained over the concentration range 10–10,000 ng/mL for teniposide, with a lower limit of quantification of 10 ng/mL. The intra‐day precision and inter‐day precision (relative standard deviation) were less than 10.23 and 13.09%, respectively. The developed method was applied for the first time to the pharmacokinetic study of teniposide in rats following a single intravenous administration of 4.5 mg/kg teniposide. Copyright © 2009 John Wiley & Sons, Ltd.     

Determination of bromazepam in human plasma by high-performance liquid chromatography with electrospray ionization tandem mass spectrometric detection: application to a bioequivalence study

A simple method using a one-step liquid-liquid extraction (LLE) followed by high-performance liquid chromatography (HPLC) with positive ion electrospray ionization tandem mass spectrometric (ESI-MS/MS) detection was developed for the determination of bromazepam in human plasma, using lorazepam as internal standard. The acquisition was performed in the multiple reaction monitoring mode, monitoring the transitions: m/z 316 > 182 for bromazepam and m/z 321 > 275 for lorazepam. The method was linear over the studied range (1-100 ng ml(-1)), with r(2) > 0.98, and the run time was 2.5 min. The intra- and inter-assay precisions were 2.7-14.6 and 4.1-17.3%, respectively and the intra- and inter-assay accuracies were 87-111 and 75.8-109.5%, respectively. The mean recovery was 73.7%, ranging from 64.5 to 79.7%. The limit of quantification was 1 ng ml(-1). At this concentration the mean intra- and inter-assay precisions were 14.6 and 7.1%, respectively, and the mean intra- and inter-assay accuracies were 102.5 and 104%, respectively. Bromazepam stability was evaluated and the results showed that the drug is stable in standard solution and in plasma samples under typical storage and processing conditions. The method was applied to a bioequivalence study in which 27 healthy adult volunteers (14 men) received single oral doses (6 mg) of reference and test bromazepam formulations, in an open, two-period, randomized, crossover protocol. The 90% confidence interval of the individual ratios (test formulation/reference formulation) for C(max) (peak plasma concentration), AUC(0-96) and AUC(0-inf) (area under the plasma concentration versus time curve from time zero to 96 h and to infinity, respectively) were within the range 80-125%, which supports the conclusion that the test formulation is bioequivalent to the reference formulation regarding the rate and extent of bromazepam absorption.     

Simultaneous analysis of ten phytohormones in Sargassum horneri by high‐performance liquid chromatography with electrospray ionization tandem mass spectrometry

Phytohormones have attracted wide attention due to their important biological functions. However, their detection is still a challenge because of their complex composition, low abundance and diverse sources. In this study, a novel method of high‐performance liquid chromatography with electrospray ionization tandem mass spectrometry was developed and validated for the simultaneous determination of ten phytohormones including indole‐3‐acetic acid, isopentenyladenine, isopentenyl adenosine, trans‐zeatin riboside, zeatin, strigolactones, abscisic acid, salicylic acid, gibberellin A3, and jasmonic acid in Sargassum horneri (S. horneri). The phytohormones were extracted from freeze‐dried S. horneri with methanol/water/methanoic acid (15:4:1, v/v/v) analyzed on a Hypersil Gold C₁₈ column and detected by electrospray ionization tandem triple quadrupole mass spectrometry in the multiple reaction monitoring mode. The experimental conditions for the extraction and analysis of phytohormones were optimized and validated in terms of reproducibility, linearity, sensitivity, recovery, accuracy, and stability. Distributions of the phytohormones in the stems, blades, and gas bladder of the S. horneri in drift, fixed, and semi‐fixed growing states were investigated for the first time. The observed contents of the phytohormones in S. horneri range from not detected to 5066.67 ng/g (fresh weight). Most phytohormones are distributed mainly in the stems of S. horneri in drift and semi‐fixed states.     

Determination of cefquinome in pig plasma and bronchoalveolar lavage fluid by high-performance liquid chromatography combined with electrospray ionization mass spectrometry

The aim of this study was to develop a rapid and sensitive method for the quantification of cefquinome in animal plasma and bronchoalveolar lavage (BAL) fluid using high-performance liquid chromatography combined with electrospray tandem mass spectrometry (LC-ESI-MS/MS). Cefadroxil is used as internal standard. For plasma, the sample preparation includes a simple deproteinization step with a Microcon filter. This allows detecting the unbound cefquinome concentration, which is correlated with the concentration in other body fluids, such as BAL fluid. To be able to detect the total plasma concentration, deproteinization with acetonitrile, followed by a back-extraction of actonitrile with dichloromethane was performed. The BAL fluid is centrifuged to precipitate floating particles. Chromatographic separation is achieved on a PLRP-S column using 0.005% formic acid and methanol as mobile phase. For plasma, good linearity was observed in the range of 5-2500 ng ml(-1) for both the unbound and total concentration. The response in BAL fluid was linear in the range of 4-1000 ng ml(-1). The limit of quantification (LOQ) was set at 5.00 ng ml(-1) for plasma and at 4.00 ng ml(-1) for BAL fluid. The limit of detection (LOD) was 3.12 ng ml(-1) and 0.41 ng ml(-1) for the unbound and total concentration in plasma, respectively, and was 1.43 ng ml(-1) for BAL fluid. The method was shown to be of use in a pharmacokinetic study in pigs, where the correlation between cefquinome concentrations in plasma and BAL fluid of pigs was studied.     

Study of the reaction products of flavonols with 2,2-diphenyl-1-picrylhydrazyl using liquid chromatography coupled with negative electrospray ionization tandem mass spectrometry

The products obtained after the reaction between flavonols and the stable free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH(*)) in both methanol and acetonitrile were characterized using liquid chromatography coupled with negative electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) and NMR spectroscopy. The flavonols studied were quercetin, kaempferol and myricetin. In methanol, two reaction products of oxidized quercetin were identified using LC/ESI-MS/MS and NMR. Quercetin was oxidized through a transfer of two H-atoms to DPPH(*) and subsequently incorporated either two CH(3)OH molecules or one CH(3)OH- and one H(2)O molecule giving the products 2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-2,3-dimethoxy-2,3-dihydrochromen-4-one and 2-(3,4-dihydroxyphenyl)-3,3,5,7-tetrahydroxy-2-methoxy-2,3-dihydrochromen-4-one, respectively. LC/ESI-MS/MS analysis revealed that in methanol, kaempferol and myricetin also gave rise to methoxylated oxidation products similar to that identified for quercetin. Kaempferol, in addition, also exhibited products where a kaempferol radical, obtained by a transfer of one H-atom to DPPH(*), reacted with CH(3)OH through the addition of CH(3)O(*), yielding two isomeric products. When the reaction took place in acetonitrile, LC/ESI-MS/MS analysis showed that both quercetin and myricetin formed stable isomeric quinone products obtained by a transfer of two H-atoms to DPPH(*). In contrast, kaempferol formed two isomeric products where a kaempferol radical reacted with H(2)O through the addition of OH(*), i.e. similar to the reaction of kaempferol radicals with CH(3)OH.     

Mass spectrometry in the study of anthocyanins and their derivatives: differentiation of Vitis vinifera and hybrid grapes by liquid chromatography/electrospray ionization mass spectrometry and tandem mass spectrometry

A mass spectrometric-based procedure for anthocyanin profiling was set up to distinguish authentic Vitis vinifera from hybrid red grapevine cultivars. 3-O-Monoglucoside and the related acetyl-, p-coumaryl- and caffeoyl-monoglucoside anthocyanins occurred only in Vitis vinifera, whereas 3,5-O-diglucoside and the substituted acetyl-, p-coumaryl-, feruloyl- and caffeoyl-diglucoside anthocyanins were the additional pigments in hybrid grapevines. The procedure was applied expressly to identify red grape cultivars based on the anthocyanin chemo-type determination. In particular, a red grape cultivar, having 3,5-O-diglucoside anthocyanins and a novel class of anthocyanin monoglucosides, such as cyanidin-3-O-, cyanidin-3-O-(6-O-acetyl)- and cyanidin-3-O-(6-O-p-coumaryl)pentoside, was classified as hybrid. A second vine cultivar, characterized exclusively by 3-O-monoglucoside anthocyanins, was included among the Vitis vinifera species. Anthocyanin profiling by mass spectrometry could represent the core of a chemotaxonomic procedure for distinguishing American and European grapevines based on the identification of post-synthetic anthocyanidin modification.     

Determination of ivermectin B(1a) in animal plasma by liquid chromatography combined with electrospray ionization mass spectrometry

A novel, sensitive and specific method for the quantitative determination of ivermectin B(1a) in animal plasma using liquid chromatography combined with positive electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) is presented. Abamectin was used as the internal standard. Extraction of the samples was performed with a deproteinization step using acetonitrile. Chromatographic separation was achieved on a Nucleosil ODS 5 microm column, using gradient elution with 0.2% (v/v) acetic acid in water and 0.2% (v/v) acetic acid in acetonitrile. The method was validated according to the requirements defined by the European Community. Calibration curves using plasma fortified between 1 and 100 ng ml(-1) showed a good linear correlation (r > or = 0.9989, goodness-of-fit coefficient < or =8.1%). The trueness at 2 and 25 ng ml(-1) (n = 6) was +4.2 and -17.1%, respectively. The trueness and between-run precision for the analysis of quality control samples at 25 ng ml(-1) was -4.0 and 11.0%, respectively (n = 16). The limit of quantification of the method was 1.0 ng ml(-1), for which the trueness and precision also fell within acceptable limits. Using a signal-to-noise ratio of 3 : 1, the limit of detection was calculated to be 0.2 ng ml(-1). The specificity was demonstrated with respect to ivermectin B(1b).The method was successfully used for the quantitative determination of ivermectin B(1a) in plasma samples from treated bovines, demonstrating the usefulness of the developed method for application in the field of pharmacokinetics.     

Rapid quantification of HIV protease inhibitors in human plasma by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry

HIV protease inhibitors are important antiretroviral drugs which have substantially reduced the morbidity and mortality associated with HIV-1 infection. Recent data have shown relationships between plasma concentrations of the protease inhibitors and clinical response, which makes therapeutic drug monitoring valuable. We have developed and validated an assay, using liquid chromatography coupled with electrospray tandem mass spectrometry (LC/MS/MS), for the routine quantification of the six licensed protease inhibitors (amprenavir, indinavir, lopinavir, nelfinavir, ritonavir and saquinavir) and the pharmacologically active nelfinavir metabolite M8 in plasma. The sample pretreatment consisted of protein precipitation with a mixture of methanol and acetronitrile using only 100 microl of plasma. Chromatographic separation was performed on an Inertsil ODS3 column (50 x 2.0 mm i.d., particle size 5 microm), with a quick stepwise gradient using an acetate buffer (pH 5) and methanol, at a flow rate of 0.5 ml min(-1). The analytical run time was 5.5 min. The use of a 96-well plate autosampler allowed batch sizes up to 150 patient samples. The triple-quadrupole mass spectrometer was operated in the positive ion mode and multiple reaction monitoring was used for drug quantification. The method was validated over the concentration ranges 0.01-10 microg ml(-1) for indinavir and saquinavir, 0.1-10 microg ml(-1) for amprenavir, 0.05-10 microg ml(-1) for nelfinavir and ritonavir, 0.1-20 microg ml(-1) for lopinavir and 0.01-5 microg ml(-1) for M8. Saquinavir-d(5) and indinavir-d(6) were used as internal standards. The coefficients of variation were always <10% for both intra-day and inter-day precisions for each compound. Mean accuracies were also between the designated limits (+/-15%). The validated concentration ranges proved to be adequate in daily practice. This robust and fast LC/MS/MS assay is now successfully applied for routine therapeutic drug monitoring and pharmacokinetic studies in our hospital.     

In vivo and in vitro metabolism of aspirin eugenol ester in dog by liquid chromatography tandem mass spectrometry

Aspirin eugenol ester (AEE) is a promising drug candidate for treatment of inflammation, pain and fever and prevention of cardiovascular diseases with fewer side effects than its precursor, aspirin. Investigation into its metabolic process in target animal species will help to illustrate its mechanism of action and to establish its residual mark compound to formulate its dosage. Six beagle dogs were orally given a dose of 20 mg kg^?1 of AEE and one dog was used to prepare blank liver microsomes. Their liver microsomes were prepared for in vitro study and their plasma and urine were collected for in vivo metabolic analysis using liquid chromatography tandem mass spectrometry. In this study we identified 10 metabolites, M1, M2, M3, M4, M5 in phase I and M6, M7, M8, M9, M10 in phase II. Based on the metabolites of AEE, the pathways of AEE metabolism in dog were demonstrated. Copyright © 2014 John Wiley & Sons, Ltd.     

液相色谱-电喷雾串联质谱法测定生姜中的215种农药残留

建立了生姜中215种农药多残留测定的液相色谱-电喷雾串联质谱(LC-ESI-MS/MS)方法。样品用1%醋酸-乙腈溶液均质提取,经Sep-Pak Vac固相萃取柱净化,乙腈-甲苯(3:1, v/v)洗脱,旋转蒸发浓缩至约0.5 mL后,于室温氮气吹干,用乙腈-水(3:2, v/v)溶解,以电喷雾电离串联质谱在正离子多反应监测(MRM)模式下进行测定。在定量限水平进行添加回收率实验,方法的回收率范围为68.1%～132.6%,其中回收率在70%～120%的占94.4%,相对标准偏差(RSD)范围为0.4%～25.0%。方法的检出限(S/N=3)和定量限(S/N=10)范围分别为0.01～70.45 μg/L和0.04～234.84 μg/L。该方法操作简便,灵敏度、准确度和精密度均符合农药多残留检测技术要求,适用于生姜中215种农药多残留的快速测定。     

Pharmacokinetics and bioavailability study of ginsenoside Rk1 in rat by liquid chromatography/electrospray ionization tandem mass spectrometry

Ginsenoside Rk1 (Rk1) exhibited various potent biological activities. However, its pharmacokinetic profile in vivo remains unclear. In the present study, a simple and sensitive liquid chromatography tandem mass spectrometry method was developed and validated for determination of Rk1 in rat plasma and applied in a pharmacokinetic study. The sample was precipitated with acetonitrile and separated on a Zorbax Eclipse XDB C18 column (50 × 2.1 mm, 1.8 μm). The mobile phase was composed of 0.1% formic acid in water and acetonitrile at a flow rate of 0.4 mL/min. Rk1 and internal standard (ginsenoside Rg3) were quantitatively monitored with precursor‐to‐product ion transitions of m/z 765.4 → 441.5 and m/z 783.5 → 621.4, respectively. The assay was linear over the concentration range of 5–1000 ng/mL (r > 0.99) with the LLOQ of 5 ng/mL. Other parameters including intra‐ and inter‐day precision and accuracy, extraction recovery and matrix effect were within the acceptable limits. The analyte was stable under the tested storage conditions. The validated method has been successfully applied to a pharmacokinetic study of Rk1 in rat plasma after intravenous (5 mg/kg) and oral (25 mg/kg, 50 mg/kg) administration. After oral administration, Rk1 could be detected in blood at 30 min and reached the highest concentration at 4.29~4.57 h. Our results demonstrated that Rk1 showed low clearance, moderate half‐life (3.09–3.40 h) and low bioavailability (2.87–4.23%). The study will provide information for the further application of Rk1.     

Quantitative profiling method for phytohormones and betaines in algae by liquid chromatography electrospray ionization tandem mass spectrometry

A liquid chromatography–tandem mass spectrometry (LC‐MS/MS) method was developed for the quantitative determination of phytohormones and betaines in algae. The results showed that phytohormones and betaines were separated with high efficiency on Hypersil Gold C₁₈ and Cnwsil SCX columns. Mass spectrometric detection was performed using positive or negative electrospray ionization in selective reaction monitoring mode (SRM). Linearity of the method was good with correlation coefficients (r²) > 0.9951 in the range of 0.005–5 mg/L. The limits of detection were from 0.004 to 0.86 µg/L and the limits of quantification were in the range from 0.01 to 2.8 µg/L for the investigated phytohormones and betaines. The obtained recoveries varied between 61.33 and 90.39%, and the relative standard deviations were <15%. Using the developed methods, seven types of phytohormones and two types of betaines in Laminaria japonica, and seven types of phytohormones and one type of betaine in Pyropia haitanensis, which were collected in Xiangshan, Zhejiang Province, China, were determined. Thus, LC‐MS/MS was demonstrated to be a powerful tool for the comprehensive analysis of phytohormones and betaines in algae, owing to its large dynamic range and excellent sensitivity. Copyright © 2013 John Wiley & Sons, Ltd.     

Characterization of [6]-gingerol metabolism in rat by liquid chromatography electrospray tandem mass spectrometry

[6]-Gingerol is a structural analog of capsaicin, an agonist of the transient receptor potential channel vanilloid 1, which is known to have therapeutic properties for the treatment of pain and inflammation. A selective and sensitive quantitative method for the determination of [6]-gingerol by HPLC-ESI/MS/MS was developed. The method consisted of a protein precipitation extraction followed by analysis using liquid chromatography electrospray tandem mass spectrometry. The chromatographic separation was achieved using a Thermo 100 × 2.1 mm C(8) column combined with an isocratic mobile phase composed of acetonitrile, water and formic acid (80:20:0.1) at a flow rate of 250 μL/min. The mass spectrometer was operating in SRM mode and an analytical range set at 20-5000 ng/mL was used to construct a calibration curve in rat plasma. The interbatch precision (%CV) and accuracy (%NOM) observed were 2.9-10.8% and 98.1-102.1% in rat plasma. Similarly, precision and accuracy in rat liver microsomal suspension were also evaluated at nominal concentrations of 1, 25 and 100 μm; the precision (%CV) was <3.4% and the accuracy (%NOM) observed ranged from 89.7 to 109.4%. An in vitro metabolic stability study using rat liver microsomes was performed to determine intrinsic clearance of [6]-gingerol. The results show slow degradation with a T(1/2) of 163 min and relatively low intrinsic clearance suggesting that phase I metabolism may not be a major contributor of the drug clearance. Further analyses were performed to characterize in vitro and in vivo metabolites. Three main phase I metabolites and four phase II metabolites were identified by HPLC-MS/MS and HPLC-MSD TOF. However, the results suggest that glucuronidation of hydroxylated [6]-gingerol is the primary metabolite excreted in rat urine.