Characterization of a new rat urinary metabolite of piperine by LC/NMR/MS studies

Potential of piperine, an active alkaloid of black and long peppers, to increase the bioavailability of drugs in humans is of great clinical significance owing to its omnipresence in food. In an attempt to further study the reported differences in its metabolism in rats and humans, a new major urinary metabolite was detected in rat urine and plasma using HPLC. The metabolite was partially purified using reverse phase column chromatography on Sephadex^®-LH 20 and characterized as 5-(3, 4-methylenedioxy phenyl)-2E,4E-pentadienoic acid-N-(3-yl propionic acid)-amide with the help of LC/NMR/positive ESI–MS studies. Complete mass fragmentation pattern could be assigned with MS/MS studies. The metabolite has a unique structure compared to the previously reported metabolites in that it retains methylenedioxy ring and conjugated double bonds while the piperidine ring is modified to form propionic acid group. Mechanism of formation of the metabolite by oxidation and cleavage of piperidine ring is proposed. Kidney appears to be the major excretion route for piperine metabolites in rats as no metabolite could be detected in feces.     

The investigation and the use of high flow column-switching LC/MS/MS as a high-throughput approach for direct plasma sample analysis of single and multiple components in pharmacokinetic studies

Recently direct plasma injection LC/MS/MS technique has been increasingly used in pharmaceutical research and development due to the demand for higher throughput of sample analyses. In this work, two on-line extraction methods including high flow LC/MS/MS and high flow column switching LC/MS/MS were investigated. The evaluations were conducted and focused on their performances with respect to peak responses, separation efficiency, and signal to-noise ratio in a multiple-component LC/MS/MS assay. Two HPLC pumps were used-with one for high flow delivery and one for gradient elution. A CTC autosampler was used to inject plasma samples. High flow LC was achieved by the use of 4 ml/min flow rate on a 1×50 mm Waters Oasis column. A 2×100 mm YMC column was coupled via a column-switching valve. The extracted analytes were analyzed in multiple-reaction-monitoring (MRM) mode using a triple quadrupole MS/MS. As a rapid and simple procedure, vortex-mixing plasma and internal standard directly in sample vials completed sample preparation. The high flow column switching method (two-column system) provided sharper peak shape than the conventional high flow method. This effect increased analyte signal-to-noise ratio and sensitivity. Narrower peak width resulted in much better separation efficiency, which was required for multiple compound (N-in-1) analysis. A 2 mm I.D. column resulted in better peak shape and resolution than using a smaller I.D. column. The selected method achieved acceptable recoveries for most of the compounds tested, and it was successfully applied to a 10-in-1 pharmacokinetic (PK) study. The results showed that the dynamic range, lower limit of quantitation, assay accuracy and precision were acceptable for all compounds. Rapid sample preparation eliminated labor intensive and time consuming processes and improved productivity. This high throughput on-line extraction high flow column switching method has been proven particularly useful for multiple component analysis in PK studies.     

A rapid and sensitive method for determination of dimethyl benzoylphenyl urea in human plasma by using LC/MS/MS

Dimethyl benzoylphenyl urea (BPU), a poorly water-soluble benzoylphenyl urea derivative, inhibits tubulin polymerization and causes microtubule depolymerization in vitro with activity against solid tumors. BPU is currently being tested in Phase I clinical trials. A rapid, sensitive and specific method using LC/MS/MS has been developed for the quantitation of BPU in human plasma to perform pharmacokinetic (PK) and pharmacodynamic (PD) studies of BPU administered orally once a week. BPU is extracted from plasma into acetonitrile-n-butylchloride and separated on a Waters X-Terra MS C18 (50 x 2.1 mm, 3.5 microm) column with acetonitrile/water mobile phase (80:20, v/v) containing 0.1% formic acid using isocratic flow at 0.15 ml/min for 5 min. The analyte of interest was monitored by tandem-mass spectrometry with electrospray positive ionization with a cone voltage 15 V for BPU and 30 V for the internal standard, paclitaxel. The detector settings allowed the monitoring of the [MH](+) ion of BPU (m/z 470.3) and the [MH](+) of internal standard paclitaxel (m/z 854.5), with subsequent monitoring of the product ions of BPU (m/z 148.0) and paclitaxel (m/z 286.1). Calibration curves were generated over the range of 0.05-10 ng/ml with values for coefficient of determination of >0.99. The values for precision and accuracy were <20 and < or =15%, respectively. Following administration of BPU 5 mg as a weekly oral dose to a patient with advanced solid tumor malignancies, the maximum plasma concentration was 6.5 ng/ml and concentrations were quantifiable up to 173 h after administration. The lower limit of quantitation (LLOQ) of 0.05 ng/ml allows for successful measurement of plasma concentrations in patients receiving therapy with BPU as a once weekly oral dose.     

Characterization and quantitative determination of impurities in piperaquine phosphate by HPLC and LC/MS/MS

Four impurities in piperaquine phosphate bulk drug substance were detected by a newly developed gradient reverse phase high performance liquid chromatographic (HPLC) method. These impurities were identified by LC/MS/MS. The structures of impurities were confirmed by spectroscopic studies (NMR and IR) conducted using synthesized authentic compounds. The synthesized reference samples of the impurity compounds were used for the quantitative HPLC determination. The system suitability of HPLC analysis established the validity of the separation. The method was validated according to ICH guidelines with respect to specificity, precision, accuracy and linearity. Forced degradation studies were also performed for piperaquine phosphate bulk drug samples to demonstrate the stability indicating power of the newly developed HPLC method.     

A rapid and sensitive LC/MS/MS assay for quantitative determination of digoxin in rat plasma

Digoxin is a cardiac glycoside that is widely used for the treatment of congestive heart failure. To evaluate pharmacokinetics of digoxin in rats, a sensitive LC/MS/MS assay was developed and validated for the determination of digoxin concentration in rat plasma. For detection, a Sciex API3000 LC/MS/MS with atmospheric pressure ionization (API) mass spectrometry turbo ion spray inlet in the positive ion-multiple reaction monitoring mode was used to monitor precursor→product ions of m/z 798.6→651.6 for digoxin and m/z 577.6→433.3 for oleandrin, the internal standard (IS). The standard curve was linear (r²≥0.999) over the digoxin concentration range of 0.1–100 ng/ml in plasma for digoxin. The mean predicted concentrations of the quality control samples deviated by <5.8% from the corresponding nominal values; the intra-assay and inter-assay precision of the assay were within 8.6% relative standard deviation. At the lower limit of quantitation (LLQ) of 0.1 ng/ml, the mean deviation of predicted concentrations from the nominal value was within 3.7%. The extraction recoveries of digoxin and internal standard were 82.7±3.9 and 105.9±2.3%, respectively. The present method was successfully applied to characterization of pharmacokinetic profiles of digoxin in rats after oral administration.     

Characterization of metabolites of xylazine produced in vivo and in vitro by LC/MS/MS and by GC/MS.

The metabolic fate of xylazine, 2-(2,6-dimethylphenylamino)-5,6-dihydro-4H-1,3-thiazine, in horses is described. The major metabolites identified in the hydrolyzed horse urine were 2-(4'-hydroxy-2',6'-dimethylphenylamino)-5,6-dihydro-4H-1,3-thiazi ne, 2-(3'-hydroxy-2',6'-dimethylphenylamino)-5,6-dihydro-4H-1,3-thiazi ne, N-(2,6-dimethylphenyl)thiourea, and 2-(2',6'-dimethylphenylamino)-4-oxo-5,6-dihydro-1,3-thiazine. These metabolites were also produced by incubating xylazine with rat liver microsomes. The major metabolite produced in vitro by rat liver preparations was found to be the ring opened N-(2,6-dimethylphenyl)thiourea. The identities of these metabolites were confirmed by spectroscopic comparisons with synthetic standards. Phenolic metabolic standards were synthesized efficiently by the use of Fenton's reagent. This reagent was used to monohydroxylate multiply substituted aromatic ring systems. LC/MS/MS, with an atmospheric pressure chemical ionization source, was found to be particularly useful in confirming the presence of phenolic metabolites in hydrolyzed equine urine and microsomal extracts. These phenolic metabolites could not be analyzed by GC/MS even after derivatization with silylating agents. The advantage of LC/MS/MS was that no or little sample preparation of urine or microsomal extract was necessary prior to the analysis. A mechanism is also proposed for the formation of the major metabolite, N-(2,6-dimethylphenyl)thiourea, from xylazine.     

液相色谱-串联质谱法测定人血浆中的米氮平及在药动学中的应用

目的:研究液相色谱-串联质谱法测定人血浆中的米氮平,并应用于生物等效性研究。方法:50μL 血浆样品经乙腈沉淀蛋白处理后,以乙腈-水-甲酸(80:20:0．2)为流动相,Zorbax SB-C_8柱分离,采用电喷零离子源,选择反应监测方式进行正离子检测。用于定量分析的离子反应分别为m／z 266→195(米氮平)和 m／z 256→167(内标,苯海拉明)。药动学参数采用非室模型计算。结果:米氮平测定方法的线性范围为0．18- 144．0 μg·L~(-1),定量下限为0．18 μg·L~(-1)。日内、日间精密度(RSD)均小于6．2％,准确度(RE)在±2．0％以内。每个样品测试时间仅为3．5 min。口服米氮平片30 mg后测得参比制剂和受试制剂的t_(max)分别为(1．4±s 0．7)h 和(1．4±0．7)h,c_(max)分别为(65±35)μg·L_(-1)和(69±35)μg·L_(-1),t_(1/2)分别为(24±6)h和(24±6)h,用梯形法计算,AUC_(0-96)分别为(814±419)μg·h·L_(-1)和(842±387)μg·h·L_(-1)。以AUC_(0-96)计算,受试制剂相对生物利用度为(102±18)％。结论:该法选择性强、灵敏度高、操作简便,适用于米氮平的制剂的生物等效性评价及临床药动学研究。     

A rapid LC/MS/MS quantitation assay for naringin and its two metabolites in rats plasma

Naringin is a flavonoid that exists in many plants and traditional Chinese medicines. In this study, a highly sensitive and specific electrospray ionization (ESI) liquid chromatography-tandem mass spectrometry (LC/MS/MS) method was developed for quantification of naringin and its two metabolites, naringenin and naringenin glucuronide. Naringin and naringenin were extracted from rat plasma with ethyl acetate, using hesperidin as an internal standard. Components in the extract were separated on a 100 mm x 2.0 mm Betabasic 5 microm C18 ODS column by isocratic elution with 70% methanol. The components were analyzed in the multiple-reaction-monitoring (MRM) mode in the precursor/product ion pair of m/z 581.3/273.4 for naringin, m/z 273.4/153.1 for naringenin and m/z 611.5/303.4 for hesperidin, respectively. Linear calibration curves were obtained in the range of 5-1000 ng/ml, using 0.1 ml rat plasma. The within-day coefficients of variation (CVs) were 3.1, 1.8 and 2.2% for naringin, 3.0, 3.3, 3.1% for naringenin at 5, 50 and 500 ng/ml (n=5). The between-day CVs were 3.4, 1.7 and 4.9% for naringin and 4.0, 3.0, 4.6% for naringenin (n=5) at 5, 50 and 500 ng/ml respectively. A formulation based on PEG400 was used and orally administered to Sprague-Dawley male rats. Plasma drug concentrations were measured by this method and the pharmacokinetics was analyzed by WinNonlin computer software. Plasma concentration-time profiles of naringin were found to increase quickly and decline rapidly within 2 h and could not be detected after 24 h. Naringenin and naringenin glucuronide occurred slower and the T(max) were about 9 and 7.5 h later, respectively.     

LC／MS／MS技术对西他沙星代谢物结构的研究

利用LC／MS／MS实现对西他沙星体内代谢物的在线鉴定,并通过定向合成的代谢物对照品,对西他沙星代谢物的结构作了进一步验证。结果鉴定出六个代谢物D2～D7。根据代谢物的结构特征证明了西他沙星在肝脏经氧化被代谢消除。     

Advanced glycation end-products/peptides: a preliminary investigation by LC and LC/MS

An investigation on AGE-peptides, originating by proteolysis of in vitro glycated proteins, was carried out by LC methods with different detection applied to the mixture produced by proteinase K digestion of in vitro glycated human serum albumin (HSA). Classical approaches, like spectroscopic (UV, fluorescence) and mass spectrometric methods (MALDI, LC/ESI/MS), show that the digestion mixture is highly complex. However, there are clearcut differences between the digestion mixtures of glycated and unglycated HSA, in the former case allowing identification of possible glycated peptides belonging to the AGE-peptide class. MS/ MS experiments on selected species seem to be promising as regards structural information.     

液相色谱-质谱联用法测定人血浆双氢青蒿素浓度

目的:建立液相色谱-质谱联用法测定健康人血浆中双氢青蒿素浓度的方法。方法:以青蒿素为内标,血浆样品采用液-液萃取法处理。用电喷雾离子化和正离子多离子反应监测方式检测双氢青蒿素。结果:该方法双氢青蒿素线性范围为1.01~2020 ng.ml-1;定量下限为1.001±0.072 ng.ml-1;方法回收率在93.0%~98.2%;批内、批间变异系数均<10%。结论:该方法准确、灵敏、特异、简便,适用于健康人血浆双氢青蒿素浓度的测定。     

高效液相色谱-质谱联用测定人血浆中辛伐他汀浓度

目的建立测定人血浆中辛伐他汀浓度的方法。方法血浆样品中加入内标,用乙醚提取,浓缩后采用高效液相色谱-质谱联用进行测定。结果血浆样品中辛伐他汀线性范围为0.1～20ng·mL-1(r=0.9999);萃取回收率为94.3%。结论本方法灵敏度高、专属性强、重现性好、准确,可用于辛伐他汀片人体药动学及生物等效性研究。     

LC-MS/MS法测定人血浆中甘草次酸及其临床药代动力学研究

目的建立人血浆中甘草次酸的LC-MS/MS测定方法,研究男性健康志愿者单剂量服用甘草酸二铵胶囊,其代谢产物甘草次酸体内药代动力学行为。方法健康男性志愿者单剂量口服甘草酸二铵胶囊150 mg,血浆样品经乙酸乙酯提取,进行LC-MS/MS分析。色谱柱为Agilent ZORBAXSB C18(3.0×100 mm,5μm),流动相为甲醇∶乙腈∶醋酸铵缓冲液(5 mmol.L-1醋酸铵,0.2%冰醋酸)(15∶60∶25,V/V/V),检测离子为m/z469.4/355.2(甘草次酸)、m/z358.9/279.9(内标泼尼松龙)。测定甘草次酸血药浓度,计算其药代动力学参数。结果在1.5～192μg.L-1内,甘草次酸与内标的峰面积比值与浓度的线性关系良好,定量限为1.5μg.L-1,提取回收率为77.14%～83.64%。人体中甘草次酸药代动力学参数:Cmax为(73.85±25.25)μg.L-1,Tmax为(11.50±3.07)h,T21β为(11.82±3.56)h,AUC0-60为(1252.49±489.06)μg.h.L-1。结论建立的LC-MS/MS分析方法准确灵敏,适于临床药代动力学研究。口服甘草酸二铵胶囊,其代谢产物甘草次酸在体内的药代动力学特点是达峰时间长,约占受试者总人数50%的药时曲线有双峰现象。     

液相色谱-质谱-质谱联用法测定人血浆中氯诺昔康液相色谱-质谱-质谱联用法测定人血浆中氯诺昔康

目的建立测定人血浆中氯诺昔康的液相色谱-质谱-质谱联用法,并用于中国受试者口服氯诺昔康的体内药代动力学研究。方法血浆样品经液-液萃取后,以甲醇-水-甲酸(80∶20∶0.5)为流动相,吡罗昔康为内标,采用Zorbax XDB-C8柱分离,通过液相色谱串联质谱,以选择反应监测(SRM)方式进行检测。定量分析离子反应分别为m/z 372→121(氯诺昔康)和m/z 332→121(吡罗昔康)。结果线性范围为2.0～1 600 μg·L^-1,定量下限为2.0 μg·L^-1。18名受试者po氯诺昔康8 mg后主要药动学参数t_1/2为(4.7±1.1) h,AUC_0-∞为(5.5±2.4) mg·h·L^-1。而另一名受试者t_1/2为105 h,AUC_0-∞为189.5 mg·h·L^-1。结论该法灵敏度高,线性范围宽,操作简便、快速,适用于临床药代动力学研究。     

Micro LC/MS in drug analysis and metabolism studies

LC/MS has become a routine research tool in some laboratories. Although no single approach to LC/MS presently is without limitations, each approach offers encouraging results and prompts continued improvements. The remaining hurdles appear to be the introduction of total LC effluent into the MS, increased sensitivity, and the ability to analyze higher molecular weight, polar molecules. The micro LC/MS results discussed in this paper offer an opportunity for significantly increased sensitivity by allowing total micro LC effluent introduction into the MS. It remains to be seen whether the development of micro LC column and equipment technology will allow practical micro LC/MS. It may someday provide improved separation of drugs, metabolites, and their conjugates from high levels of endogenous materials in reasonable time periods. Currently, the impressive efficiencies demonstrated for micro LC columns involve mixtures containing low molecular weight solutes that are perhaps more amenable to GC analysis. The analyst routinely involved with problem solving must deal with complex mixtures composed of components with large concentration differences. The future of micro LC, and hence micro LC/MS, will depend upon how well the technique helps solve problems. We believe the future is bright for micro LC/MS. The techniques may require some fine tuning of operating procedures, just as with capillary GC/MS, but certainly the potential for increased efficiency and sensitivity is worth the effort. Currently we are testing a new micro LC/MS DLI probe wherein the exit of the micro column is within 1 cm of the MS ion source [82]. The micro LC column is contained within the DLI probe and should offer the lowest dead volume and the least extracolumn effects yet achieved by LC/MS. Initial testing of this new probe is under way, and experimental results will be reported subsequently. The analytical potential of micro LC/MS is receiving considerable interest. Practical micro LC performance can provide increased capabilities to all types of LC/MS reported to date, and perhaps offer new insight into alternative methods of LC/MS not yet reported. We look forward to learning of these breakthroughs as they become available.     

液相色谱-质谱-质谱联用法测定人血浆中阿德福韦的浓度

目的:建立快速、灵敏的液相色谱-质谱-质谱联用(LC-MS-MS)法测定人血浆中阿德福韦的浓度,并研究其在中国男性健康志愿者人体内的药动学。方法:以乙腈-0.1%甲酸为流动相,采用梯度洗脱,以5-溴尿嘧啶为内标。血浆样品经乙腈沉淀蛋白后用二氯甲烷提取,经ZorbaxXDB-C8柱分离后,通过电喷雾离子化三重四极杆串联质谱仪,以多反应检测(MRM)方式进行测定。结果:阿德福韦的线性范围为0.167～83.3μg.L-1(r=0.9989),平均相对回收率在88.3%～110.0%之间,日内、日间精密度的RSD均小于12%,阿德福韦的定量下限为0.167μg.L-1。结论:该方法具有快速、准确、灵敏等优点,适用于阿德福韦的药动学研究。     

Simple, sensitive and rapid LC-MS/MS method for the quantitation of cerivastatin in human plasma--application to pharmacokinetic studies

A simple and sensitive liquid chromatography-tandem mass spectrometry method was developed and validated for estimation of cerivastatin (I) in human plasma, a potent hydroxy-methylglutaryl-coenzyme A reductase inhibitor. The analyte and internal standard (atorvastatin, II) were extracted by liquid/liquid extraction with diethyl ether/dichloromethane (70/30, v/v). The chromatographic separation was performed on reverse phase Xterra ODS column with a mobile phase of water/acetonitrile (30/70, v/v) with 0.03% formic acid. The protonated analyte was quantitated in positive ionization by multiple reaction monitoring with a mass spectrometer. The mass transitions m/z 460.4 --> 356.3 and 559.2 --> 440.3 were used to measure I and II, respectively. The lower limit of quantitation was 10pg/mL with a relative standard deviation of less than 15%. Acceptable precision and accuracy were obtained for concentrations over the calibration curve ranges (0.01-10ng/mL). Sample analysis time of 2min for each sample made it possible to analyze a throughput of more than 400 human plasma samples per day. The assay can be used to analyze human plasma samples to support phase I and II clinical studies.     

Automated system for on-line desorption of dried blood spots applied to LC/MS/MS pharmacokinetic study of flurbiprofen and its metabolite

This paper illustrates the development of an automated system for the on-line bioanalysis of dried blood spots (on-line DBS). In this way, a prototype was designed for integration into a conventional LC/MS/MS, allowing the successive extraction of 30 DBS toward the analytical system without any sample pretreatment.