High-throughput determination of atrasentan in human plasma by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry

Atrasentan (A-147627) is an endothelin antagonist receptor being developed at Abbott Laboratories for the treatment of prostate cancer. A quick and sensitive method for the determination of atrasentan in human plasma has been developed and validated using high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry. A dual-column, single mass spectrometer system is used to provide a reliable and routine means to increase sample throughput. The analytical method involves liquid-liquid extraction and internal standard (A-166790). The plasma samples and internal standard are acidified with 0.3 M hydrochloric acid prior to being extracted into 1:1 (v/v) hexanes--methyl t-butyl ether. The organic extract was evaporated to dryness using heated nitrogen stream and reconstituted with mobile phase. Atrasentan and internal standard were separated with no interference in a Zorbax SB-C(18) analytical column with 2.1 x 50 mm, 5 microm, and a Zorbax C(8) guard column using a mobile phase consisting of 50:50 (v:v) acetonitrile--0.05 M ammonium acetate, pH 4.5, at a flow rate of 0.30 mL/min to provide 4 min chromatograms. For a 250 microL plasma sample volume, the limit of quantitation was approximately 0.3 ng/mL. The calibration was linear from 0.30 to 98.0 ng/mL (r(2) > 0.995). A significant advantage of the method is the ability to employ parallel HPLC separations with detection by a single MS/MS system to provide sensitivity and selectivity sufficient to achieve robust analytical results with a lower limit of quantitation of 0.30 ng/mL and high throughput.     

Determination of lovastatin in human plasma by ultra-performance liquid chromatography/electrospray ionization tandem mass spectrometry

A fast and sensitive ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed for the determination of lovastatin in human plasma. With simvastatin as internal standard, sample pretreatment involved one-step extraction with n-hexane-methylene dichloride-isopropanol (20:10:1, v/v/v) of 0.5 mL plasma. Chromatographic separation was carried out on an Acquity UPLC BEH C(18) column with mobile phase consisting of acetonitrile-water (containing 5 mmol/L ammonium acetate; 85:15, v/v) at a flow-rate of 0.35 mL/min. The detection was performed on a triple-quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) via electrospray ionization source with positive mode. The analysis time was shorter than 1.7 min per sample. The standard curve was linear (r2>or=0.99) over the concentration range 0.025-50.0 ng/mL with a lower limit of quantification of 0.025 ng/mL. The intra- and inter-day precision values were below 11% and the accuracy (relative error) was within 6.0% at three quality control levels. This is the first method of MS with MRM coupled to UPLC for the determination of lovastatin, which showed great advantages of high sensitivity, selectivity and high sample throughput. It was fully validated and successfully applied to the pharmacokinetic study of lovastatin tablets in healthy Chinese male volunteers after oral administration.     

Determination of aripiprazole in rat plasma and brain using ultra-performance liquid chromatography/electrospray ionization tandem mass spectrometry

Aripiprazole is an important antipsychotic drug. A simple, sensitive and rapid ultra‐performance liquid chromatography/electrospray ionization tandem mass spectrometry (UPLC‐ESI‐MS/MS) method was developed and validated for the simultaneous quantification of this compound in rat plasma and brain homogenate. The analyte was extracted from rat plasma and brain homogenate using a weak cation exchange mixed‐mode resin‐based solid phase extraction. The compound was separated on an Agilent Eclipse Plus C₁₈ (2.1 × 50 mm, 1.8 µm) column using a mobile phase of (A) 0.1% formic acid aqueous and (B) acetonitrile with gradient elution. The analyte was detected in positive ion mode using multiple reaction monitoring. The method was validated and the specificity, linearity, limit of quantitation (LOQ), precision, accuracy, recoveries and stability were determined. The LOQ was 0.5 ng/mL for aripiprazole in plasma and 1.5 ng/g in brain tissue. The MS response was linear over the concentration range 0.5–100 ng/mL for aripiprazole in plasma and 1.5–300 ng/g in brain tissue. The precision and accuracy for intra‐day and inter‐day were better than 14%. The relative and absolute recoveries were above 72% and the matrix effects were low. This validated method was successfully used to quantify the rat plasma and brain tissue concentrations of the analyte following chronic treatment with aripiprazole. Copyright © 2012 John Wiley & Sons, Ltd.     

Simultaneous determination of amino acids and neurotransmitters in plasma samples from schizophrenic patients by hydrophilic interaction liquid chromatography with tandem mass spectrometry

A sensitive, reproducible, and rapid method was developed for the simultaneous determination of underivatized amino acids (aspartate, serine, glycine, alanine, methionine, leucine, tyrosine, and tryptophan) and neurotransmitters (glutamate and γ‐aminobutyric acid) in plasma samples using hydrophilic interaction liquid chromatography coupled to triple quadrupole tandem mass spectrometry. The plasma concentrations of amino acids and neurotransmitters obtained from 35 schizophrenic patients in treatment with clozapine (27 patients) and olanzapine (eight patients) were compared with those obtained from 38 healthy volunteers to monitor the effectiveness of treatment. The chromatographic conditions separated ten target compounds within 3 min. This method presented linear ranges that varied from (lower limit of quantification: 9.7–13.3 nmol/mL) to (upper limit of quantification: 19.4–800 nmol/mL), intra‐ and interassay precision with coefficients of variation lower than 10%, and relative standard error values of the accuracy ranged from –2.1 to 9.9%. The proposed method appropriately determines amino acids and neurotransmitters in plasma from schizophrenic patients. Compared with the control group (healthy volunteers), the plasma levels of methionine in schizophrenic patients treated with olanzapine are statistically significantly higher. Moreover, schizophrenic patients treated with clozapine tend to have increased plasma levels of glutamate.     

Ultra‐performance liquid chromatography‐tandem mass spectrometry method for the determination of febuxostat in dog plasma and its application to a pharmacokinetic study

A rapid, sensitive and selective ultra‐performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method was developed and validated for the determination of febuxostat in dog plasma. Using paclitaxel as an internal standard (IS), a simple liquid–liquid extraction method with ethyl acetate was adopted for plasma sample pretreatment. Separation was carried out on an Acquity UPLC BEH C₁₈ column with a mobile phase consisting of acetonitrile and water (containing 0.2% formic acid). The assay was linear in the concentration ranged from 5 to 5000 ng/mL with a lower limit of quantification of 5 ng/mL for febuxostat. The single run analysis was as short as 2.0 min. Finally, the developed method was successfully applied to the pharmacokinetic study of febuxostat tablets following oral administration at a single dose of 40 mg in beagle dogs. Copyright © 2012 John Wiley & Sons, Ltd.     

Quantitative analysis of docetaxel in human plasma using liquid chromatography coupled with tandem mass spectrometry

An assay for the quantitative determination of docetaxel in human plasma is described. Docetaxel was extracted from the matrix using liquid-liquid extraction with ter-butylmethylether, followed by high-performance liquid chromatographic analysis using an alkaline eluent. Paclitaxel was used as internal standard. Positive ionization electrospray tandem mass spectrometry was performed for selective and sensitive detection. The method was validated according to the FDA guidelines on bioanalytical method validation. The validated range for docetaxel was from 0.25--1000 ng/mL using 200 microL plasma aliquots. The method requires only a limited volume (200 microL) of human plasma and the method can be applied in studies requiring a low lower limit of quantitation of 0.25 ng/mL. The assay was applied successfully in several clinical and pharmacological studies with docetaxel.     

Acrylamide determination in atmospheric particulate matter by high-performance liquid chromatography/electrospray ionisation tandem mass spectrometry

A method has been developed for the determination of acrylamide at the picogram per cubic metre level in particulate-phase outdoor aerosol using high-performance liquid chromatography with triple quadrupole tandem mass spectrometric detection. Acrylamide was identified by positive ion electrospray mass spectrometry using m/z 72.00/54.90 as monitoring ion transition. The limit of detection, defined as three times the standard deviation of the procedural blanks, was 0.4?pg?m^?3 (173?pg absolute amount injected); the repeatability was 8% (evaluated as the relative standard deviation of five consecutive measurements on cleaned quartz fibre filters of acrylamide standard spikes) and the recovery was 52?±?4%. The accuracy of the method (evaluated as relative error) has been estimated to be ?2%. This methodology was used to determine acrylamide concentrations in particulate-phase outdoor aerosol in the Venice Lagoon with concentrations ranging between 0.4 and 12.9?pg?m^?3 with an average value of 3.1?pg?m^?3.     

4-Methylamino antipyrine determination in human plasma by high-performance liquid chromatography tandem mass spectrometry

In the present study, a simple and rapid method for metamizole metabolite 4-methylamino antipyrine (MAA) determination in human plasma was developed, validated and successfully applied to a clinical trial. Chromatographic separation was achieved in HILIC mode on a YMC-Pack SIL column (100 × 2.0 mm; S-5 μm, 30 nm), with a mobile phase consisting of acetonitrile, water and formic acid. Protein precipitation of a small plasma volume using acetonitrile was selected for sample preparation. The multiple reaction monitoring transitions in the positive ionization mode were m/z 218.2 → 56.2 for MAA and m/z 221.2 → 56.2 for MAA-d3 (IS, internal standard). Concentration levels of MAA calibration standards were in the range of 0.100–20 μg/ml. Metamizole conversion into MAA in both water and organic media was investigated, and the level of the conversion in commercially available injection solutions was estimated.     

超高效液相色谱-三重四极杆质谱法同时检测人尿中12种单羟基多环芳烃代谢物

建立了高效、准确的同时测定人体尿样中多种多环芳烃代谢物的方法。取10.0 mL尿液,酶解,经固相萃取净化浓缩、0.2 μm滤膜过滤后,以Acquity UPLC^®HSS T3（100 mm×2.1 mm,1.8 μm）为分析柱,甲醇和水为流动相,采用负离子电喷雾多反应监测模式检测样品中多环芳烃代谢物的含量。12种多环芳烃代谢物在0.04~20.0 μg/L范围内线性关系良好,相关系数>0.99,方法检出限为0.01~0.41 μg/L,平均回收率为80.0%~105%,批内RSD为1.21%~9.12%,批间RSD为4.43%~19.7%。应用该方法对淮河流域某区域的100份人体尿样进行了检测,多环芳烃代谢物的检出率为98%。该方法操作简单,灵敏度高,选择性好,可同时检测尿中12种羟基多环芳烃代谢物。     

Determination of acyclovir in horse plasma and body fluids by high-performance liquid chromatography combined with fluorescence detection and heated electrospray ionization tandem mass spectrometry

Two methods are presented for the determination of 'respectively' the plasma protein unbound and total concentration of acyclovir in horse plasma and body fluids: first, a liquid-liquid extraction was performed on plasma, combined with HPLC-fluorescence detection for the total plasma concentration; second a more sensitive method using high-performance liquid chromatography combined with heated electrospray ionization tandem mass spectrometry (LC-HESI-MS/MS) was described for plasma and for body fluids analysis. To obtain the unbound concentration of acyclovir in plasma, a simple deproteinization step using a Microcon filter was performed. Ganciclovir was used as an internal standard. Analysis was carried out on an Inertsil 5 ODS-3 column for the HPLC-fluorescence method. For the LC-HESI-MS/MS method a PLRP-S column was used. The limit of quantification (LOQ) for the total concentration was set at 50 and 2 ng mL(-1) for the HPLC-fluorescence method and the LC-HESI-MS/MS method, respectively. The limit of quantification for the unbound concentration was set at 5 ng mL(-1) and at 2 ng mL(-1) for body fluids. The methods were successfully used to perform pharmacokinetic and clinical studies in horses after intravenous and oral dosage of acyclovir and its prodrug valacyclovir.     

Improved extraction and identification by ultra performance liquid chromatography tandem mass spectrometry of phenolic compounds in burdock leaves

The simultaneous ultrasonic and microwave assisted extraction (UMAE) technique was first employed to obtain phenolics. The effects of UMAE variables including extraction time, microwave power, and solvent to solid radio on the yield of phenolics were investigated. The optimized conditions were as follows: solvent to solid ratio was 20:1 (ml/g), extraction time was 30 s, microwave power was 500 W and two times of extraction. Moreover, the phenolic yield of UMAE was higher than that by maceration, indicating a significant reduction of extraction time and an improvement of efficiency. The phenomenon is related to the strong disruption of leaf tissue structure by microwave induced expansion and ultrasonic shaking, which had been observed with the scanning electron microscopy. The phenolic compositions of the extract was then identified by ultra performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS), 10 compounds had been characterized, providing a more complete identification of phenolic compounds in burdock leaves than previously reported. The occurrence of benzoic acid and p-coumaric acid is reported for the first time. This study suggests that UMAE is a good alternative for the extraction of phenolics, with a great potential for industrial application. Also, UMAE provides a new sample preparation technique for characterization of the phenolic compounds from plants.     

固相萃取-高效液相色谱/串联质谱法测定食醋中的3种甜味剂

建立了食醋中3种甜味剂的固相萃取-高效液相色谱/串联质谱分析方法。样品经酸性水稀释,弱阴离子固相萃取柱净化,Pursuit C18色谱柱分离,10 mmol/L醋酸铵(含0.1%氨水)和乙腈为流动相梯度洗脱,电喷雾负离子模式下多反应监测(MRM)模式检测。糖精钠、安赛蜜、甜蜜素的定量限分别为10、5、5 μg/kg,回收率为72.1%～96.8%,相对标准偏差小于15%。该方法准确、灵敏度高,可用于食醋中甜味剂的定性定量检验。     

Simultaneous determination of prochlorperazine and its metabolites in human plasma using isocratic liquid chromatography tandem mass spectrometry

Oral prochlorperazine (PCZ), an antiemetic, undergoes extensive first-pass metabolism. The study developed a simultaneous analytical method for PCZ and its major metabolites, prochlorperazine sulfoxide (PCZSO), N-demethylprochlorperazine (NDPCZ) and 7-hydroxyprochlorperazine (PCZOH), in human plasma using an isocratic liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. Deproteinized plasma specimens were separated using a 3 μm particle size octadecylsilyl column, and the run time was 10 min. The calibration curves were linear over the concentration ranges of 0.01-40 μg/L for PCZ, NDPCZ and PCZOH, and 0.05-80 μg/L for PCZSO. The intra- and inter-assay precisions and accuracies were within 7.0 and 99-104% and within 9.0 and 99-105%, respectively. The lower limits of quantification in human plasma were 10 ng/L for PCZ, NDPCZ and PCZOH, and 50 ng/L for PCZSO. The validated method was applied to the determination of plasma samples in 37 cancer patients receiving PCZ. Large interindividual variations were observed in plasma concentrations of PCZ, PCZSO, NDPCZ and PCZOH (relative standard deviation, 89.4, 88.7, 86.4 and 78.2%, respectively). In conclusion, this simultaneous LC-MS/MS method with acceptable analytical performance can be helpful for evaluating the pharmacokinetics of PCZ, including the determination of its metabolites in cancer patients and in clinical research.     

Determination of bencycloquidium bromide in dog plasma by liquid chromatography with electrospray ionization tandem mass spectrometry

A rapid, selective and sensitive liquid chromatography/tandem mass spectrometry (LC‐MS/MS) method was developed and validated for determining bencycloquidium bromide (BCQB) in beagle dog plasma. The plasma sample was deproteinized with methanol which contained l‐ethyl‐bencycloquidium bromide as internal standard, and supernantant was assayed by LC‐MS/MS. The chromatographic separation was performed on a Phenomenex C₁₈ column (100 × 2.0 mm, i.d., 3.0 μm) with a gradient programme mobile phase consisting of methanol and ammonium acetate (5 mm) containing 0.15% acetic acid and at a flow rate of 0.3 mL/min. Electrospray ionization in positive ion mode and selective reaction monitoring was used for the quantification of BCQB with a monitored transitions m/z 330.2 → 142.1 for BCQB and m/z 344.2 → 126.2 for IS. Validation results indicated that the lower limit of quantification was 0.05 ng/mL and the assay exhibited a linear range of 0.05–10.0 ng/mL and gave a correlation coefficient of 0.9998. The intra‐ and inter‐run precisions of the assay were 1.7–4.6 and 3.2–15.6%, respectively, and the intra‐ and inter‐day accuracies were ?8.8 to 1.1 and ?5.0 to 4.6%, respectively. The developed method was applied for the pharmacokinetic study of BCQB in beagle dogs following a single intranasal dose. Copyright © 2009 John Wiley & Sons, Ltd.     

Quantitative determination of buprenorphine,naloxone and their metabolites in rat plasma using hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry

A rapid and sensitive LC–MS/MS method was developed and validated for the simultaneous determination of buprenorphine and its three metabolites (buprenorphine glucuronide, norbuprenorphine and norbuprenorphine glucuronide) as well as naloxone and its metabolite naloxone glucuronide in the rat plasma. A hydrophilic interaction chromatography column and a mobile phase containing acetonitrile and ammonium formate buffer (pH 3.5) were used for the chromatographic separation. Mass spectrometric detection was achieved by an electrospray ionization source in the positive mode coupled to a triple quadrupole mass analyzer. The calibration curves for the six analytes displayed good linearity over the concentration range 1.0 or 5.0–1000 ng/mL. The intra and inter‐day precision (CV) ranged from 2.68 to 16.4% and from 9.02 to 14.5%, respectively. The intra‐ and inter‐day accuracy (bias) ranged from −14.2 to 15.2% and from −9.00 to 4.80%, respectively. The extraction recoveries for all the analytes ranged from 55 to 86.9%. The LC–MS/MS method was successfully applied to a pharmacokinetic study of buprenorphine–naloxone combination in rats.     

Elucidating the structure of carbon nanoparticles by ultra-performance liquid chromatography coupled with electrospray ionisation quadrupole time-of-flight tandem mass spectrometry

A fast and accurate ultra-performance liquid chromatography coupled with electrospray ionisation quadrupole time-of-flight tandem mass spectrometry (UPLC-ESI-Q-TOF-MS/MS) method was developed for the separation and structural elucidation of fluorescent carbon nanoparticles (CNP). The CNP was synthesised from microwave-assisted pyrolysis of citric acid (CA) and 1,2-ethylenediamine (EDA). By using UPLC separation, the CNP product was well separated into ten fractions within 4.0 min. Based on high-accuracy MS and MS/MS analyses, the CNP species were revealed to display six kinds of chemical formulas, including (C₁₀H₂₀N₄O₅)_n, (C₈H₁₂N₂O₅)_n, (C₁₆H₂₂N₄O₉)_n, (C₆H₈O₇)_n, (C₁₄H₁₈N₂O₁₁)_n, and (C₁₄H₁₆N₂O₁₀)_n. In particular, our study revealed for the first time that the CNP species exist as supramolecular clusters with their individual monomers units linked together through non-covalent bonding forces. These findings clearly indicated the usefulness of UPLC-ESI-Q-TOF-MS/MS in identifying the chemical composition of CNP product. It is anticipated that our proposed methodology can be applied to study the structure-property relationships of CNP, facilitating in the production of CNP with desirable spectral features.     

高效液相色谱-荧光检测法分析麦类中麦角克列斯汀碱

提出了一种采用高效液相色谱-荧光检测法(HPLC-FLD)测定麦类样品中麦角克列斯汀碱的方法。麦类样品经V(乙腈)∶V(0.1 mol/L乙酸铵缓冲溶液)=1∶4提取,以C18小柱净化,C18色谱柱(4.6×250 mm,5μm)分离,V(水)∶V(乙腈)=3∶2作流动相,流速1.0 mL/min,以HPLC-FLD定量测定。标准工作溶液浓度在1.0~50.0μg/L范围内,与峰面积成良好的线性关系,线性相关系数0.9999,样品在10.0、50.0、250.0μg/kg添加水平的回收率为76%~85%,相对标准偏差(RSD)为6.6%~8.8%(n=8),方法检测限为5.0μg/kg(S/N10)。     

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.     

A simplified method for the determination of total homocysteine in plasma by electrospray tandem mass spectrometry

Hyperhomocysteinemia is a risk factor for different diseases. Several methods have been developed to analyze homocysteine and the immunometric ones, although expensive, they are in widespread use. A rapid LC‐MS/MS method for homocysteine assay has been developed for the application of large clinical chemistry routines. Selected reaction monitoring was performed through the transitions m/z 136.0→90.1 for homocysteine and m/z 140.0→94.0 for the internal standard. ESI was used to generate [H+] adduct ions. Chromatographic isocratic separation was achieved using a strong cation exchange column. The mobile phase was methanol/water (20:80 v/v, containing 0.1% formic acid and 1.5 mmol/L ammonium formate in the water phase) at a flow rate of 0.250 mL/min (35°C). Samples treatment consisted in the reduction with DTT and deproteinization with methanol. Recovery, linearity, LOD, LOQ and total imprecision were evaluated to validate the method. Homocysteine values on 100 serum samples were compared with those obtained by HPLC and immunometric methods. The method is robust, selective and precise in the whole range of values studied. Moreover, low reagent cost and easiness of sample treatment make this method useful, not only for research, but also for routine work.     

Validated ultra-performance liquid chromatography tandem mass spectrometry method for the determination of pramipexole in human plasma

A sensitive and high throughput ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS-MS) method has been developed for the determination of pramipexole, a dopamine agonist, in human plasma. Sample preparation involved liquid-liquid extraction of pramipexole and ranitidine as the internal standard (IS) in ethyl acetate from 100 μL human plasma. The chromatographic separation is achieved on a Waters Acquity UPLC BEH C18 (100 mm × 2.1 mm, 1.7 μm) analytical column using an isocratic mobile phase, consisting of 10 mM ammonium formate (pH 7.50)-acetonitrile (15:85, v/v), at a flow-rate of 0.5 mL/min. The precursor → product ion transition for pramipexole (m/z 212.1 → 153.0) and IS (m/z 315.0 → 176.1) were monitored on a triple quadrupole mass spectrometer, operating in the multiple reaction monitoring (MRM) and positive ion mode. The method was validated over a wide dynamic concentration range of 20-4020 pg/mL. Matrix effect is assessed by post-column infusion experiment and the process efficiency were 91.9% and 85.7% for pramipexole and IS, respectively. The method is rugged and rapid with a total run time of 1.5 min and is applied to a bioequivalence study of 0.25 mg PPX tablet formulation in 30 healthy Indian male subjects under fasting condition.