Direct-injection high performance liquid chromatography ion trap mass spectrometry for the quantitative determination of olanzapine,clozapine and N-desmethylclozapine in human plasma

A specific and sensitive direct-injection high performance liquid chromatography electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS) method has been developed for the rapid identification and quantitative determination of olanzapine, clozapine, and N-desmethylclozapine in human plasma. After the addition of the internal standard dibenzepin and dilution with 0.1% formic acid, plasma samples were injected into the LC/MS/MS system. Proteins and other large biomolecules were removed during an online sample cleanup using an extraction column (1 x 50 mm i.d., 30 microm) with a 100% aqueous mobile phase at a flow rate of 4 mL/min. The extraction column was subsequently brought inline with the analytical column by automatic valve switching. Analytes were separated on a 5 microm Symmetry C18 (Waters) analytical column (3.0 x 150 mm) with a mobile phase of acetonitrile/0.1% formic acid (20:80, v/v) at a flow rate of 0.5 mL/min. The total analysis time was 6 min per sample. The inter- and intra-assay coefficients of variation for all compounds were <11%. By eliminating the need for extensive sample preparation, the proposed method offers very large savings in total analysis time.     

Short communication performance of octadecylsilylated monolithic silica capillary columns of 530 microm inner diameter in HPLC

Monolithic silica capillary columns were successfully prepared in a fused silica capillary of 530 microm inner diameter and evaluated in HPLC after octadecylsilylation (ODS). Their efficiency and permeability were compared with those of columns pakked with 5-microm and 3-microm ODS-silica particles. The monolithic silica columns having different domain sizes (combined size of through-pore and skeleton) showed 2.5-4.0-times higher permeability (K= 5.2-8.4 x 10(-14) m2) than capillary columns packed with 3-mm particles, while giving similar column efficiency. The monolithic silica capillary columns gave a plate height of about 11-13 microm, or 11 200-13 400 theoretical plates/150 mm column length, in 80% methanol at a linear mobile phase velocity of 1.0 mm/s. The monolithic column having a smaller domain size showed higher column efficiency and higher pressure drop, although the monolithic column with a larger domain size showed better overall column performance, or smaller separation impedance (E value). The larger-diameter (530 microm id) monolithic silica capillary column afforded a good peak shape in gradient elution of proteins at a flow rate of up to 100 microL/min and an injection volume of up to 10 microL.     

Ultra-high capacity liquid chromatography chip/quadrupole time-of-flight mass spectrometry for pharmaceutical analysis

Nanoflow liquid chromatography/mass spectrometry (nano-LC/MS) has attracted increasing interest in virtue of high sensitivity, low sample consumption, and minimal matrix effect. In this work a HPLC-Chip/quadrupole time-of-flight (Q-TOF) MS device with a new ultra-high capacity small molecule chip (UHC-Chip) which features a 500 nL enrichment column and a 150 mm × 75 μm analytical column, was evaluated with a drug mixture covering a wide range of polarities. Excellent chromatographic precision with 0.1-0.5% RSD for retention time and 1.7-9.0% RSD for peak area, low limit of detection, good chip-to-chip reproducibility and linearity were obtained by using this UHC-Chip. Compared with the standard HPLC-Chip with 40 nL trapping column, the UHC-Chip showed higher enrichment capability and hence gave a higher response in signal detection. Additionally, 4-30 times increase in sensitivity was obtained compared with conventional LC/MS, which indicated that UHC-Chip/MS was a valuable tool for the quantitative analysis of low level impurities and degradation products in pharmaceuticals. Moreover, satisfactory results obtained from trace drug analysis of serum samples further proved its practicality and potential for use in drug testing and development.     

Effects of column length, particle size, gradient length and flow rate on peak capacity of nano-scale liquid chromatography for peptide separations

The effects of the column length, the particle size, the gradient length and the flow rate of a nanoLC system on peptide peak capacity were investigated and compared. Columns packed with 1.7 microm and 3 microm C(18) materials into pieces of 75 microm capillary tubing of various lengths were tested with different gradient lengths and flow rates. While increasing the length of a column packed with the 1.7 microm material helped improve peptide peak capacity at the whole range of the tested gradient lengths (24-432 min), little improvement in peak capacity was observed with the increase of the length of a column packed with the 3 microm material unless a gradient longer than 50 min was carried out. Up to 30% of peak capacity increase was observed when a column's length is doubled, with little reduction in the throughput. In most cases, more than 50% of the increase in peak capacity was obtained with the reduction in the particle size from 3 microm to 1.7 microm. With the same backpressure generated, a shorter 1.7-microm-particle column outperformed a longer column packed with the 3 microm material. In a flow rate range of 100-700 nl/min, increasing the flow rate improved peak capacity for columns packed with 1.7 microm and 3 microm materials.     

Quantitative determination of coenyzme Q10 by liquid chromatography and liquid chromatography/mass spectrometry in dairy products

The dietary sources of CoQ10 and the evaluation of CoQ10 in dairy products were characterized. For quantitation of CoQ10 in food samples, 2 liquid chromatography (LC) methods with UV and mass spectrometry (MS) detections were developed. LC with UV detection was performed at 25 degrees C on a Hyperclone ODS 5 microm 150 x 4.6 mm column with mobile phase consisting of methanol-ethanol-2-propanol (70 + 15 + 15, v/v/v). Flow rate was 1.0 mL/min. Retention time of CoQ10 was 10.9 +/- 0.1 min. The method was sensitive [limit of detection (LOD) = 0.2 mg/kg], reproducible [relative standard deviation (RSD) = 3:0%), and linear up to 25 mg/kg (R > 0.999). LC/MS analysis was performed on a LUNA C18 3 microm, 150 x 4.6 mm column, using mobile phase consisting of ethanol-dioxane-acetic acid (9 + 1 + 0.01, v/v/v), flow rate was 0.6 mL/min, and the retention time of CoQ10 was 4.1 +/- 0.1 min. Identification and quantitation were performed with a Finnigan-LCQ mass detector in positive atmospheric pressure chemical ionization mode. Mass spectra were obtained in selected-ion monitoring mode; molecular mass (M+H)+ m/z 863.4 +/- 1 was used for quantitative determination. MS detection is more sensitive than UV detection (LOD = 0.1 mg/kg), less reproducible (RSD = 4.0%), and linear in selected range. Analytical recoveries are 75-90% and depend on the ratio between the amount of fat in the matrix and the concentration of CoQ10 in the sample. Some soybean milk products were analyzed together with different cow, goat, and sheep milk products. Concentrations obtained with LC and LC/MS were compared with a few accessible results available from the literature. Concentrations varied from 0 ppm in soybean milk to nearly 2 ppm in fresh milk from local farms.     

万古霉素键合手性固定相高效液相色谱法直接分离泰妥拉唑对映体

利用反相高效液相色谱法在大环抗生素类手性固定相万古霉素键合手性固定相(Chirobiotic V)上直接分离了泰妥拉唑对映体。考察了缓冲溶液的种类、浓度和pH值,有机改性剂的种类和浓度,柱长和柱温等对手性分离的影响。优化后的色谱条件为:Chirobiotic V色谱柱(150 mm×4.6 mm,5 μm),流动相为0.02 mol/L 醋酸铵缓冲液(pH 6.0)-四氢呋喃(体积比为93∶7),流速为0.5 mL/min,柱温为20 ℃,检测波长为306 nm。在此条件下泰妥拉唑对映体达到了基线分离,分离度达1.68;对映体保留时间的相对标准偏差分别为0.48%和0.49%(n=6),峰面积的相对标准偏差分别为0.45%和0.55%(n=6)。所建立的手性分离方法具有简便快速及重复性好等优点。     

高效液相色谱法同时测定诺诺感冒片中扑尔敏、扑热息痛、盐酸伪麻黄碱的含量

 采用反相高效液相色谱法 ,在C18柱上以V(甲醇 )∶V(水 ) =2 5∶75的溶液为流动相 (内含 0 .0 5mol/L磷酸二氢钠 ) ,检测波长为 2 0 5nm ,同时分离测定诺诺感冒片中扑尔敏、扑热息痛、盐酸伪麻黄碱的含量。扑尔敏、扑热息痛和盐酸伪麻黄碱的检出限分别为 1.16mg/L ,0 .15mg/L和 1.82mg/L ,其相应的回收率分别为 98.35 % (n =5 ,RSD =1.6 0 % ) ,10 1.16 % (n =5 ,RSD =1.5 0 % )和 98.5 0 % (n =5 ,RSD =1.5 9% )。方法简便、快速 ,重现性好 ,适用于诺诺感冒片的质量检验分析。     

高效液相色谱-质谱法分析菊芋叶中的绿原酸类化合物

建立了菊芋叶中绿原酸类化合物的高效液相色谱-紫外检测-质谱(HPLC-UV-MS)定性分析方法。液相色谱条件:Inertsil ODS-3色谱柱(250 mm×4.6 mm,5 μm);甲醇和水(含1%乙酸)梯度洗脱,流量1.0 mL/min;柱温35 ℃;检测波长327 nm。质谱条件:Thermo公司TSQ三级四极杆质谱仪;电喷雾电离(ESI)接口;负离子检出模式。采用该方法得到了菊芋叶提取物的紫外检测的色谱图、负离子监测的总离子流图以及相应色谱峰的紫外光谱图和一级、二级质谱图,对其进行解析,鉴别出菊芋叶中的7个绿原酸类成分。该方法简便、快速、灵敏度高,可以很好地对菊芋叶中的绿原酸类化合物进行定性分析。     

替考拉宁键合手性固定相高效液相色谱法直接拆分泮托拉唑钠对映体

以替考拉宁为手性选择剂制备了大环抗生素类手性固定相替考拉宁键合手性固定相(T-CSP),建立了T-CSP反相液相色谱直接拆分泮托拉唑钠对映体的方法。考察了流动相中有机改性剂的种类和比例、柱温以及流动相流速对拆分泮托拉唑钠对映体的影响。研究发现,用甲醇作有机改性剂比乙腈更有利于对映体的分离;在研究的温度范围内,随着柱温的升高,对映体的保留时间缩短,同时分离因子和分离度降低;在一定范围内降低流速有利于对映体的分离。采用T-CSP色谱柱(150 mm×4.6 mm i.d.,5 μm),以甲醇-水(体积比为35∶65)为流动相,在流速0.6 mL/min、检测波长290 nm、柱温20 ℃的条件下,泮托拉唑钠对映体获得了近于基线的分离,所建立的方法具有简便快速及重复性好等优点。     

Microchip-based capillary electrochromatography using packed beds

Integration of a packed column onto a microchip for performance of capillary electrochromatography (CEC) is described. The quartz device incorporated a cross-injector, and a double weir trapping design for formation of 1, 2 and 5 mm long CEC columns. Three fluorescent dyes were baseline-resolved with plate numbers of 330,(330,000 plates/m; height equivalent to a theoretical plate, H = 3.0 microm) for BODIPY 493/503, 360 (360,000 plates/m; H = 2.8 microm) for rhodamine 123 and 244 (244,000 plates/m; H = 4.1 microm) for acridine orange (AO) with 500 V applied on a 1 mm long column. The 2 mm column yielded approximately 1.8 times more theoretical plates than did the 1 mm column, when operated at the same flow rate. Van Deemter plots were obtained for the three column lengths, showing increased plate height for the 5 mm length. A 2 mm column gave peak height and area relative standard deviation (RSD) values of 2.5 and 3.3%, respectively, as averages for the three dyes (n = 15). The RSD for the dye retention times was 1% (n = 6) over one day, and 3% (n = 30) over five days. Indirect fluorescence detection of thiourea and of amino acids was possible using a neutral indicator dye (BODIPY 493/503), with a detection limit of 10 microM for amino acids.     

高效液相色谱法测定血清中头孢噻肟浓度

用固相萃取法处理样品,以扑热息痛为内标物、甲醇－醋酸钠／醋酸缓冲溶液作流动相,采用反相高效液相色谱法测定血清中头孢噻肟的浓度。头孢噻肟和内标物的平均回收率分别为９６．７％和９７．７％,头孢噻肟血清浓度在１０ｍｇ／Ｌ至１５０ｍｇ／Ｌ范围内有良好线性关系,最低检测浓度为２ｍｇ／Ｌ,日内和日间相对标准偏差分别在３．０％和４．１％以内。结果表明方法准确、简便。     

Validation and application of a high-performance liquid chromatography-tandem mass spectrometric method for simultaneous quantification of lopinavir and ritonavir in human plasma using semi-automated 96-well liquid-liquid extraction

Kaletra is an important antiretroviral drug, which has been developed by Abbott Laboratories. It is composed of lopinavir (low-pin-a-veer) and ritonavir (ri-toe-na-veer). Both have been proved to be human immunodeficiency virus (HIV) protease inhibitors and have substantially reduced the morbidity and mortality associated with HIV-1 infection. We have developed and validated an assay, using liquid chromatography coupled with atmospheric pressure chemical ionization tandem mass spectrometry (LC/MS/MS), for the routine quantification of lopinavir and ritonavir in human plasma, in which lopinavir and ritonavir can be simultaneously analyzed with high throughput. The sample preparation consisted of liquid-liquid extraction with a mixture of hexane: ethyl acetate (1:1, v/v), using 100 microL of plasma. Chromatographic separation was performed on a Waters Symmetry C(18) column (150 mm x 3.9 mm, particle size 5 microm) with reverse-phase isocratic using mobile phase of 70:30 (v/v) acetonitrile: 2 mM ammonium acetate aqueous solution containing 0.01% formic acid (v/v) at a flow rate of 1.0 mL/min. A Waters symmetry C(18) guard column (20 mm x 3.9 mm, particle size 5 microm) was connected prior to the analytical column, and a guard column back wash was performed to reduce the analytical column contamination using a mixture of tetrahydrofuran (THF), methanol and water (45:45:10, v/v/v). The analytical run was 4 min. The use of a 96-well plate autosampler allowed a batch size up to 73 study samples. A triple-quadrupole mass spectrometer was operated in a positive ion mode and multiple reaction monitoring (MRM) was used for drug quantification. The method was validated over the concentration ranges of 19-5,300 ng/mL for lopinavir and 11-3,100 ng/mL for ritonavir. A-86093 was used as an internal standard (I.S.). The relative standard deviation (RSD) were <6% for both lopinavir and ritonavir. Mean accuracies were between the designed limits (+/-15%). The robust and rapid LC/MS/MS assay has been successfully applied for routine assay to support bioavailability, bioequivalence, and pharmacokinetics studies.     

Fast, low-pressure gas chromatography triple quadrupole tandem mass spectrometry for analysis of 150 pesticide residues in fruits and vegetables

We developed and evaluated a new method of low-pressure gas chromatography-tandem mass spectrometry (LP-GC/MS-MS) using a triple quadrupole instrument for fast analysis of 150 relevant pesticides in four representative fruits and vegetables. This LP-GC (vacuum outlet) approach entails coupling a 10 m, 0.53 mm i.d., 1 μm film analytical column between the MS transfer line and a 3 m, 0.15 mm i.d. capillary at the inlet. The MS creates a vacuum in the 10 m analytical column, which reduces the viscosity of the He carrier gas and thereby shifts the optimal flow rate to greater velocity. By taking advantage of the H(2)-like properties of He under vacuum, the short analytical column, a rapid oven temperature ramp rate, and the high selectivity and sensitivity of MS/MS, 150 pesticides were separated in <6.5 min. The 2.5 ms dwell time and 1 ms interscan delay of the MS/MS instrument were critical for achieving >8 data points across the 2-3 s wide peaks. To keep dwell and cycle times constant across all peaks, each segment consisted of 30 analytes (60 transitions). For assessment, we injected extracts of spiked broccoli, cantaloupe, lemon, and sweet potato from the updated QuEChERS sample preparation method. Average recoveries (n=72) were 70-120% for 144 of the pesticides, and reproducibilities were <20% RSD for all but 4 analytes. Also, detection limits were <5 ng/g for all but a few pesticides, depending on the matrix. In addition to high quality performance, the method gave excellent reliability and high sample throughput, including easy peak integration to obtain rapid results.     

Hydrophilic interaction 10 microm I.D. porous layer open tubular columns for ultratrace glycan analysis by liquid chromatography-mass spectrometry

The sensitivity of glycan analysis using nano-liquid chromatography interfaced with electrospray ionization mass spectrometry (ESI-MS) increases with the decrease of the mobile phase flow rate, accompanied by reduced ion suppression. In this study, we describe the preparation and performance of high efficiency 10 microm I.D. amine-bonded poly(vinylbenzyl chloride-divinylbenzene) hydrophilic interaction (HILIC) porous layer open tubular (PLOT) columns operated at 20 nL/min for the separation and analysis of glycan mixtures. HILIC-PLOT columns with a uniform porous polymer layer were reproducibly prepared ( approximately 4% RSD in retention time from column-to-column) via in situ polymerization, followed by one step modification with ethylenediamine. When coupled on-line with negative ESI-MS, low detection limits (0.3fmol) for a 3-sialyl-tetrasaccharide were achieved using a 2.5mx10 microm I.D. HILIC-PLOT column. A dextran ladder standard was used to evaluate the performance of the column, and high efficiency separation was achieved with detection of the dextrans up to G22 from approximately 50 fmol amounts injected. As an example of the high sensitivity of the column, MS(6) characterization of glycan structures was possible from the injection of 10 fmol of a neutral and sialylated glycan. As another example of high sensitivity LC-MS analysis of 3 ng of a PNGase F digest of ovalbumin allowed 28 N-linked glycans to be confidently identified from a single analysis. High quality MS/MS spectra for each ovalbumin glycan were acquired and manually interpreted for structure analysis. The HILIC-PLOT column is a very promising approach for LC-MS analysis of glycans at the ultratrace level.     

A novel interface for variable flow nanoscale LC/MS/MS for improved proteome coverage

A variable flow "peak trapping" liquid chromatography (LC) interface has been developed for the coupling of nanoscale LC to electrospray ionization mass spectrometry (ESI-MS). The presented peak trapping LC interface allows for the extended analysis time of co-eluting compounds and has been employed for the identification of proteins via tandem mass spectrometry (MS/MS). The variable flow process can be controlled either manually or in a completely automated manner where the mass spectrometer status determines the status of the variable flow interface. When the mass spectrometer operates in MS survey mode, the interface is operated in a so-called "high-flow" mode. Alternatively, the interface is operated in a "low-flow" mode during MS/MS analysis. In the "high-flow" mode of the variable flow process the column flow rate is typically around 200 nL/min, whereas in the "low-flow" mode the column effluent is introduced into the source of the mass spectrometer at 25 nL/min. In addition to the flow reduction during MS/MS analysis, the gradient is paused to preserve the peptide separation on the analytical nanoscale LC column. The performance of the variable flow nanoscale LC/MS/MS interface is demonstrated by the automated analysis of standard peptide mixtures and protein digests utilizing variable flow, data-dependent scanning MS/MS techniques, and automated database searching.     

High-performance liquid chromatographic method for the determination and pharmacokinetic study of wogonoside in rat serum after oral administration of traditional Chinese medicinal preparation Huang-Lian-Jie-Du decoction

A high-performance liquid chromatographic method for the determination of wogonoside in plasma of rats administrated orally with the traditional Chinese medicinal preparation Huang-Lian-Jie-Du decoction was developed. Sample preparation was carried out by protein precipitation with a mixture of acetonitrile and methanol (1:1, v/v). The extracted sample was separated on a Hypersil C(18) (150 x 5 mm i.d., 5 microm) analytical column by linear gradient elution using 0.05% (v/v) phosphoric acid (containing 5 mm sodium dihydrogen phosphate) and acetonitrile as mobile phase at a flow rate of 1.5 mL/min. The eluate was detected using a UV detector at 276 nm. The assay was linear over the range 0.109-7.0 microg/mL (R(2) = 0.9999, n = 5). Mean recovery was determined as 98.39%. Intra- and inter-day precisions (RSD) were < or =7.59%. The limit of quantitation was 0.109 microg/mL. After validation, the HPLC method developed was applied to investigate the preliminary pharmacokinetics of wogonoside in rat after oral administration of Huang-Lian-Jie-Du decoction.     

A robust micro-electrospray ionization technique for high-throughput liquid chromatography/mass spectrometry proteomics using a sanded metal needle as an emitter

A 60 microm internal diameter (i.d.) stainless-steel needle was adapted to the orthogonal ESI probe ( microESI) of a commercial ion trap mass spectrometer, and used for capillary liquid chromatography/tandem mass spectrometry (LC/MS/MS) protein identification experiments. The modification allows for the use of nitrogen sheath gas which helps in the nebulization at LC flow rates exceeding 500 nL/min and eliminates problems caused by liquid junctions commonly used to initiate nanospray ionization (NSI). A comparison is made between the performance of a 75 microm i.d. column with a 15 microm pulled glass tip using a liquid junction, and that of a 150 microm i.d. column using the new microESI device. The combination of the 150 microm i.d. column and microESI gave sensitivity close to that of NSI (250 attomoles horse heart myoglobin digest on column), and proved to be more robust than the standard pulled glass tips of similar i.d. No evidence of metal needle catalyzed oxidation of methionine was observed during analysis of the tetrapeptide MRFA under a range of test conditions. Phosphorylated peptides in a beta-casein tryptic digest were also successfully identified using the microESI interface with a steel needle. In addition it was found that a mild sanding of the metal needle tip improved spray performance.     

反相离子对高效液相色谱法测定铁强化酱油中的乙二胺四乙酸铁钠

建立了用反相离子对高效液相色谱法分离和测定铁强化酱油中的铁营养强化剂乙二胺四乙酸铁钠(NaFeEDTA)的方法。样品经甲醇沉淀后,以Zorbax C8 色谱柱(150 mm×4.6 mm i.d.,5 μm)进行分离,以含12.5%甲醇、0.13%四丁基氢氧化铵(TBAOH)和0.052%甲酸的水溶液（pH 3.5）作为流动相,流速为1.00 mL/min,检测波长为254 nm,整个分离过程在30 min内完成。考察了NaFeEDTA在不同品牌酱油中的回收率,其中NaFeEDTA的添加量为0.50~4.00 g/L时,其回收率为94.15%~101.5%。对NaFeEDTA添加量为2.00 g/L的铁强化酱油样品重复测定6次,其峰面积的相对标准偏差为0.89%。NaFeEDTA标准溶液的最低检测限为0.03 mg/L。本方法简单、快速、重现性好,可用于铁强化酱油中NaFeEDTA含量的检测。     

Determination of Abacavir in maternal plasma, amniotic fluid, fetal and placental tissues by a polarity switching liquid chromatography/tandem mass spectrometry method

A rapid and efficient high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) method for the determination of concentrations of the carbocyclic nucleoside antiviral Abacavir in maternal rat plasma, amniotic fluid, placental and fetal tissue samples has been developed and validated. All tissue samples were homogenized in water prior to analysis and all samples were prepared by acetonitrile precipitation followed by dilution with HPLC-grade water. Separation of the analyte and internal standard from the matrices was achieved on a C(8) analytical column (2.1 x 150 mm, 5 microm particle size). The mobile phase consisted of 10 mM ammonium acetate/acetonitrile using a gradient method at a flow rate of 0.25 mL/min for all matrices. The method yields retention times of approximately 3.4 and 5.1 min for the internal standard (Azidouridine) and Abacavir, respectively. For all matrices the limit of detection was approximately 1 ng/ml. Recoveries from the different matrices ranged from 53-87% for Abacavir and from 69-84% for Azidouridine. Within- and between-run precision (%RSD) and accuracy (%Error) were under 15% for all matrices.     

朝鲜淫羊藿的色谱指纹图谱及液相色谱/电喷雾串联质谱分析

建立了长白山区朝鲜淫羊藿药材的高效液相色谱指纹图谱的分析方法. 确定了18批朝鲜淫羊藿药材的13个共有峰, 该指纹图谱的精密度、稳定性和重现性的相对标准偏差均低于3.0%. 结合液相色谱/电喷雾串联质谱对特征峰进行了结构确认, 并根据电喷雾串联质谱数据推测了13个特征化合物的结构. 结果表明采用高效液相色谱与质谱联用技术对朝鲜淫羊藿色谱指纹图谱中的特征峰进行结构确认, 使其色谱指纹图谱的特征性更强, 更适合于药材质量的鉴别与评价.