Development of a novel method for triterpenoidal saponins in rat plasma by solid-phase extraction and high-performance liquid chromatography tandem mass spectrometry

A novel method using high-performance liquid chromatography tandem mass spectrometry (HPLC–MS/MS) has been developed for the quantification of four triterpenoidal saponins (anemoside B4, pulsatilloside B, anemoside A3, and 23-hydroxybetulinic acid) in rat plasma following solid-phase extraction (SPE). The optimized procedure utilized off-line extraction of the analytes from plasma using polymeric (Strata-X) SPE cartridges. Detection and quantitation were performed by MS/MS using electrospray ionization (ESI) and multiple reaction monitoring (MRM) in a novel multiswitching monitoring mode. The analytes and internal standard (scutellarin) were analyzed using a Sapphire C18 column (250 × 4.6 mm, 5 μm) with a linear gradient elution. The mass transition ion pairs of the triterpenoidal saponins were executed as follows: m/z 1219.7/749.4 for anemoside B4, m/z 819.4/347.2 for pulsatilloside B, m/z 749.6/471.2 for anemoside A3, m/z 471.4/471.4 for 23-hydroxybetulinic acid, and m/z 461.1/285.0 for the internal standard. The specificity, linearity, accuracy, precision, recovery, matrix effect, and stabilities were validated for all analytes in the plasma samples. In conclusion, the validation results demonstrate that this method is robust and specific. This validated method is a novel technique for sample preparation and quantitation and was successfully applied to estimate the pharmacokinetics of triterpenoidal saponins.     

百合总皂苷提取工艺及抗抑郁活性研究

本文研究百合总皂苷最佳提取工艺,继而采用AB-8大孔吸附树脂富集纯化百合总皂苷,并测试其抗抑郁活性。采用正交实验,考察乙醇浓度、提取次数、固液比及提取时间四个因素对该工艺的影响;采用小鼠悬尾实验和小鼠强迫游泳实验评价百合总皂苷的抗抑郁活性。确立了百合总皂苷的最佳提取工艺为:提取时间3h,乙醇浓度80%,固液比1∶10,提取次数为3次。在最佳提取工艺条件下,总皂苷提取率为1.24%。经过AB-8大孔树脂富集纯化总皂苷的含量达到62%以上。药理实验表明,百合总皂苷中剂量、小剂量能明显缩短小鼠悬尾的不动时间和游泳时间(P<0.05或者P<0.01),百合富集纯化的总皂苷部位具有较好的抗抑郁活性。     

O‐glycoside sequence of pentacyclic triterpene saponins from Phytolacca bogotensis using HPLC‐ESI/multi‐stage tandem mass spectrometry

Introduction – The lack of pharmacopoeial methodologies for the quality control of plants used for therapeutic purposes is a huge problem that impacts directly upon public health. In the case of saponins, their great structural complexity, weak glycoside bonds and high polarity hinder their identification by conventional techniques. Objective – To apply high‐performance liquid chromatography–electrospray tandem mass spectrometry (HPLC‐ESI/MSⁿ) to identify the O‐glycoside sequence of saponins from the roots of Phytolacca bogotensis. Methodology – Saponins were isolated by preparative HPLC and characterised by NMR spectroscopic experiments. Collision‐induced dissociation (CID) of isolated saponins was performed producing typical degradation reactions that can be associated with several glycosidic bonds as empirical criteria. A method using solid‐phase extraction (SPE) and HPLC/ESI‐MSⁿ for the characterisation of saponins and identification of novel molecules is described. Results – Three saponins reported for the first time in P. bogotensis were isolated and characterised by NMR spectroscopy. Characteristic cross ring cleavage reactions have been used as empirical criteria for the characterisation of the glycosidic bonds most frequently reported for Phytolacca saponins. One new saponin was proposed on the basis of empirical criteria, and other five saponins were identified for the first time for P. bogotensis using HPLC‐ESI/MSⁿ. Conclusion – Electrospray ionisation in combination with tandem mass spectrometry has been established as a powerful tool for the profiling of saponins from roots of P. bogotensis. CID proved to be a useful tool for the characterisation and identification of known and novel saponins from the plant family Phytolaccaceae and can be used for quality control purposes of crude plant extracts. Copyright © 2009 John Wiley & Sons, Ltd.     

Isolation of four high-purity dammarane saponins from extract of Panax notoginseng by centrifugal partition chromatography coupled with evaporative light scattering detection in one operation

Introduction – Centrifugal partition chromatography (CPC), as a continuous liquid–liquid partition chromatography with no solid support matrix, combined with evaporative light scattering detection (ELSD) was employed for systematic separation and purification of weak‐chromophoric saponins from a highly valued and important traditional Chinese herbal medicine, Panax notoginseng. Objective – To separate and isolate high‐purity saponins from extract of Panax notoginseng using CPC‐ELSD with a simple and low toxicity solvent system. Methodology – Samples were preparaed by extracting the root material with acetone, treated with n‐butanol and then freeze‐dried. CPC‐ELSD was applied in the separation and detection of notoginsenoside and ginsenosides from extract of Panax notoginseng using a solvent system composed of ethyl acetate–n‐butanol–water (1:1:2, v/v/v). The saponins were analysed and identified by their retention time with high‐performance liquid chromatography (HPLC) coupled with ELSD, as well as electrospray ionisation tandem mass spectrometry (ESI‐MSⁿ ) in the negative and positive ion modes with the authentic standards. Results – A total of 9.6 mg of notoginsenoside R₁, 67.8 mg of ginsenoside Rg₁, 2.3 mg of Re and 286.5 mg of Rb₁ were purified from 487.2 mg of n‐butanol extract of P. notoginseng. The purities of obtained saponins in a single run were assessed to be over 98% by HPLC‐ELSD. Conclusion – CPC‐ELSD was proved to be a very fast and efficient tool for separation of high‐purity dammarane saponins. Copyright © 2011 John Wiley & Sons, Ltd.     

茉莉酸甲酯对牛膝生长及主要药用成分积累的影响

不同浓度的外源茉莉酸甲酯(Me JA)对牛膝生长及其主要药用成分齐墩果酸和蜕皮甾酮均有不同的影响。该文采用不同浓度的Me JA分别浸种处理牛膝种子:0(对照)、0.1 mmol·L-1(T1)、0.15 mmol·L-1(T2)、0.2 mmol·L-1(T3)、0.25 mmol·L-1(T4),对照(CK)以同体积的蒸馏水浸泡。取浸种处理后生长培养60 d的牛膝植株,测定牛膝的株高、根长、地上及地下部分的生物量;取牛膝的叶及根,HPLC法测定其齐墩果酸和蜕皮甾酮的含量,研究Me JA对牛膝生长及主要药用成分齐墩果酸和蜕皮甾酮积累的影响。结果表明:0.15 mmol·L-1Me JA浸种处理,对牛膝生长及生物量的促进作用最佳,其株高、根长、地上部分鲜重及根鲜重分别比对照显著升高43.9%、38.7%、26.4%、64.0%(P0.05);0.15 mmol·L-1Me JA处理,对牛膝的根和叶中齐墩果酸的积累作用最佳,分别比对照组显著增加了114.3%和60%(P0.05);0.25 mmol·L-1Me JA处理,对牛膝根中蜕皮甾酮的积累最佳,比对照高出90.3%(P0.05),却不利于根和叶中齐墩果酸的积累,并可抑制叶中蜕皮甾酮的形成。说明0.15 mmol·L-1Me JA浸种处理对牛膝的生长及其根和叶中齐墩果酸的积累作用最佳,并能显著促进根中蜕皮甾酮的积累,有利于牛膝药材产量和品质的提高。     

基于转录组学的白扁豆总皂苷抑制前列腺癌细胞系PC-3细胞生长的机制研究

目的 白扁豆总皂苷是中药白扁豆经过提取分离纯化步骤制备得到,关于白扁豆的总皂苷成分如何影响前列腺癌细胞系PC-3细胞的生长情况缺少研究。因此,有必要探讨白扁豆总皂苷对前列腺癌细胞系PC-3细胞生长的机制研究。方法 本研究采用CCK8方法检测不同浓度的白扁豆总皂苷对前列腺癌细胞系PC-3细胞生长的影响。利用转录组学分析白扁豆总皂苷抑制前列腺癌细胞系PC-3细胞生长的分子机制,并且进一步通过实时定量PCR（qRT-PCR）和蛋白质免疫印迹（Western blot）实验对相关差异基因的表达进行验证。利用Western blot和CCK8检测白扁豆总皂苷处理过表达醛脱氢酶7家族成员A1（ALDH7A1）的PC-3细胞存活率。结果 随着白扁豆总皂苷浓度升高,前列腺癌细胞PC-3的存活率显著下降,白扁豆总皂苷的IC₅₀值为1 086 mg/L。转录组学测序结果显示,与对照组相比,白扁豆总皂苷处理的细胞中有2 360个差异表达基因,其中1 982个基因上调,378个基因下调。基因功能注释（GO）结果显示,差异表达基因显著富集到与有丝分裂纺锤体检查点（mitotic spindle checkpoint）、纺锤体组装检查点（spindle assembly checkpoint）等一系列跟癌症的发生发展密切相关的生物学过程。此外,基因组京都百科全书（KEGG）分析结果也显示,差异表达基因富集在肿瘤代谢等信号通路。进一步对其中的差异基因进行验证,结果显示,与对照组相比,白扁豆总皂苷处理的前列腺癌细胞中ALDH7A1、甘氨酸C-乙酰转移酶（GCAT）和磷酸甘油酸变位酶家族成员4（PGAM4）的蛋白质表达水平明显降低（P<0.05）,而二甲基甘氨酸脱氢酶（DMGDH）和胱硫醚β合成酶样（CBSL）的蛋白质表达水平显著升高（P<0.001）。体外细胞实验结果表明,白扁豆总皂苷通过下调前列腺癌细胞中ALDH7A1的表达抑制PC-3细胞生长。结论 白扁豆总皂苷可能通过下调ALDH7A1表达从而在体外抑制前列腺癌细胞的生长。     

黄芪三七复方两种不同制备方法活性组分与功效对比研究

摘要 目的：对比分析黄芪三七复方经本草提取机器快速制备的汤液（HSQ）与传统煎煮汤液（HS）的活性组分、免疫功能及抗氧化活性，探索传统中药复方汤药制备新技术。方法：采用粒度分析仪对黄芪和三七复方本草提取液HSQ和传统汤液HS提取物进行了粒度分析，并用可见分光光度法对其总多糖和总皂苷活性成分的含量进行了分析。腹腔注射抗肿瘤药物阿霉素和灌胃地塞米松分别在NIH小鼠上诱导两种免疫功能紊乱的动物模型。同时，灌胃给予HSQ和HS 连续4周，并通过计算其胸腺和脾脏指数来评估其对免疫功能障碍的调节作用。采用可见光分光光度法测定HSQ和HS与NIH小鼠脑组织匀浆液体外孵育后MDA（丙二醛）含量的变化。结果：与传统汤液HS相比，黄芪三七复方本草提取液HSQ的颗粒平均粒径约为HS的32.7%，尺寸更小、更均一，而且HSQ中总多糖与总皂苷含量显著增加，分别提升27%和50%。在抑制抗肿瘤药物阿霉素引起的小鼠胸腺萎缩，以及激素地塞米松引起的脾脏萎缩等免疫系统紊乱方面也具有更好的活性。另外，黄芪三七复方本草提取液HSQ在体外对MDA生成的抑制率比传统汤液HS高47%，其抗氧化活性更优。结论：相对传统汤液HS，黄芪三七复方本草提取液HSQ拥有更好的微纳形态，具有更高水平的总皂苷与总多糖等活性成分，并且表现出更好的免疫调控与抗氧化活性，该研究对传统汤液制备技术的创新具有较好的示范研究与探索意义。     

巨尾桉叶中皂苷的提取分离及其抗氧化活性研究

开展了从巨尾桉叶中提取皂苷的工艺优化研究.考察了温度、时间,溶剂用量对皂苷得率的影响,并对粗提物进行纯化.结果表明,巨尾桉叶皂苷的优化条件是:提取温度61.8℃,提取时间5.02h,溶剂与巨尾桉叶的液料比为20.09∶1,皂苷的提取得率(g/g)为5.51％.提取物经大孔树脂-丙酮沉淀联用分离法纯化后皂苷含量由16.22％提高到50.35％.对粗提物、大孔树脂纯化物和大孔树脂吸附-丙酮沉淀纯化物进行抗氧化活性研究.结果表明,它们对DPPH自由基的最大清除率分别为83.02％、84.20％和85.62％,对过氧化氢的最大清除率分别为80.10％、55.02％和48.21％,对超氧阴离子自由基的最大清除率分别为16.62％、13.43％和10.01％.     

吉林长白山刺五加总皂苷超声提取工艺研究

以吉林长白山野生刺五加果实为原料,采用正交试验设计通过香草醛—高氯酸显色法对不同超声处理条件下刺五加果实粗提物中总皂甙得率进行测定,优化野生刺五加果实总皂甙超声提取工艺.结果表明:超声提取吉林长白山野生刺五加果实总皂甙的最佳工艺条件为料液比1∶20、乙醇体积分数70％、超声波功率450W、超声提取时间50 min,测得刺五加粗提物中总皂甙含量为3.57 mg/g,该提取方法简便、快捷,可用于长白山野生刺五加果实总皂甙提取和测定.     

Analysis of bisdesmosidic saponins in Saponaria vaccaria L. by HPLC-PAD-MS: identification of new quillaic acid and gypsogenin 3-O-trisaccharides

A high-performance liquid chromatographic method using photodiode array and single quadrupole electrospray mass detection for analysis and profiling of bisdesmosidic saponins in Saponaria vaccaria seed was developed. Profiles of seed extract from three different plant sources were obtained and found to contain the same saponins, albeit in different proportions. Several known saponins were identified by selected ion extraction of quasi-molecular ions from the total ion chromatogram and confirmed by their mass spectra. Application of high cone voltages afforded mass spectra containing key diagnostic fragments and relatively strong singly charged quasi-molecular ions. In addition to previously identified saponins, several new quillaic acid and gypsogenin bisdesmosides could be detected via mass spectral analysis. Five of these were tentatively identified as pentose homologues of known saponins, having an added xylosyl residue linked to the 3-O-glucuronyl group (1 --> 3). The stereochemistry and identity of the xylosyl linkage in the new saponins was determined by chemical means. Previously reported vaccaric or segetalic acid-type bisdesmosides could not be detected in any of the extracts.     

MORPHOMETRIC ANALYSIS OF POLLEN FROM FOUR SPECIES OF AMBROSIA (COMPOSITAE)

A morphometric approach proved useful for characterizing pollen from the Ambrosia species that are responsible for most ragweed hayfever and for correctly identifying individual pollen grains. Pollen was harvested from 4 species (A. trifida, A. artemisiifolia, A. bidentata, A. psilostachya) grown in Champaign County, Illinois, and observed with a scanning electron microscope. Quantitative measurements of 6 different exine characters were made on micrographs of 96 pollen grains from each species. Discriminant analysis based on these characters was used to obtain a population centroid for each species and indicated that these centroids were distinctly different. This analysis also included a species classification for individual pollen grains and indicated probability of species membership. About 86% of all individuals were correctly classified when the 4 species were included, with a range from 80% (A. artemisiifolia) to 95% (A. psilostachya). When three species (A. trifida, A. artemisiifolia, A. bidentata) were analyzed, about 87% of the individuals were correctly classified, and almost all individuals (97%) were classified correctly when only A. trifida and A. artemisiifolia were included. The validity of the derived discriminant functions was also tested by treating herbarium specimens as unknowns. The species and percentages of these individuals correctly classified were A. trifida (100%), A. artemisiifolia (58%), A. bidentata (100%), and A. psilostachya (31%). The classification results using three species were A. trifida (100%), A. artemisiifolia (75%), and A. bidentata (100%); and using two species were A. trifida (100%) and A. artemisiifolia (92%). Hence, the disciminant functions do seem valid for 3 species, but further investigation of A. psilostachya is needed. This type of approach may be useful in pollen analysis, especially where these species significantly contributed to aeroallergens.     

Biotransformation of steriodal saponins in Dioscorea zingiberensis C. H. Wright to diosgenin by Trichoderma harzianum

Diosgenin is an important starting material in the steroidal hormone industry. Traditionally, diosgenin is mainly produced by acid hydrolysis of Dioscorea zingiberensis C. H. Wright (DZW) tubers. This method yields numerous byproducts that can cause serious pollution. In this study, diosgenin was obtained by biotransformation of steroidal saponins in DZW afforded by Trichoderma harzianum CGMCC 2979. The medium was optimized for maximum diosgenin production. The addition of phosphate buffer, surfactant Tween-85, and Fe²⁺ increased the yield of diosgenin by 50.28%, 33.35%, and 22.07%, respectively. The optimum medium obtained by response surface methodology was composed of 60 mmol l⁻¹ phosphate buffer, 0.07% (w/v) Tween-85, and 0.93 mmol l⁻¹ Fe²⁺. Under these conditions, a maximum diosgenin yield of 30.05 ± 0.59 mg g⁻¹ was achieved, which was slightly higher than that obtained from traditional acid hydrolysis. By hydrolyzing the un-transformed steroidal saponins after biotransformation, the total diosgenin yield increased by 35% compared to traditional method. Moreover, chemical oxygen demand and residual reduced sugar in the wastewater produced by this integrated process were only 3.72% and 0.3%, respectively, that of the traditional acid hydrolysis method.     

Isolation of chlorophylls and carotenoids from freshwater algae using different extraction methods

Usually marine algae are an excellent source of pigments for different commercial sectors. Freshwater macroalgae can be exploited as a good source of biologically active compounds provided an appropriate extraction method is developed. The efficiency of four methods, like microwave‐assisted (MAE), ultrasound‐assisted extraction (UAE), supercritical fluid extraction (SFE) with ethanol as a co‐solvent, as well as conventional Soxhlet extraction were studied in the same conditions (time, solvent and temperature) for the recovery of chlorophylls and carotenoids from three freshwater green algae species: Cladophora glomerata, Cladophora rivularis and Ulva flexuosa. UV‐Vis spectrophotometry was used to determine chlorophyll a, chlorophyll b and total carotenoid content in obtained extracts. The results of this study showed that the advantages of novel extraction techniques (MAE and UAE) include higher yield and, in consequence, lower costs compared to traditional solvent extraction techniques. These methods were much more efficient in freshwater green algae pigment recovery than the classic Soxhlet extraction as well as SFE.     

Liquid chromatographic determination of total celiprolol or (S)-celiprolol and (R)-celiprolol simultaneously in human plasma

A method has been developed for the determination of total celiprolol (sum of enantiomers) or the enantiomers (R)-celiprolol and (S)-celiprolol in plasma by high-performance liquid chromatography with UV and fluorescence detection. After extraction from alkalinized plasma with methyl-tert-butyl ether and back-extraction into 0.01 M HCl (for total celiprolol determination) or after evaporation of the organic phase and derivatisation with R(−)-1-(1-naphthyl)ethyl isocyanate (enantiomer determination), total celiprolol or its diastereomeric derivatives were chromatographed on a reversed-phase HPLC column with a mixture of acetonitrile and phosphate buffer pH 3.5 (+0.05% triethylamine). Acebutolol was used as internal standard. Linearity was obtained in the range of 5 to 2000 ng/ml for total and 2.5 to 500 ng/ml for enantiomer determination. Intra-day and inter-day variation was lower than 10%. The method can be applied for analysis of plasma samples obtained from patients treated with oral racemic celiprolol doses.     

Three-liquid-phase extraction of diosgenin and steroidal saponins from fermentation of Dioscorea zingibernsis C. H. Wright

Diosgenin is an important starting material in the steroidal hormone industry. The yield of diosgenin obtained from the fermentation of Dioscorea zingibernsis C. H. Wright (DZW) by Trichoderma harzianum is higher than that typically obtained from acid hydrolysis. In this paper, the extraction of steroids in the culture broth was studied. A novel three-liquid-phase system (TLPS) consisted of petroleum ether, ethanol, ammonium sulphate and water was used to separate diosgenin and steroidal saponins in the culture broth. The partition behaviors of various steroidal saponins, diosgenin and glucose were investigated. From this, an optimized TLPS was obtained, which composed of 30% ethanol (w/w), 17% (NH₄)₂SO₄ (w/w) and 40% (w/w) petroleum ether. In the optimized TLPS, almost all of the diosgenin was extracted into the top phase giving a recovery of 97.24%, whereas the steroidal saponins were mainly extracted into the middle phase, with recoveries of zingibernsis newsaponin, deltonin and diosgenin-diglucoside reaching almost 100%. The recoveries of trillin and diosgenin-triglucoside were 96.03% and 98.82%, respectively. Glucose tended to remain in the bottom phase, giving a recovery of 72.01%. The three-liquid-phase extraction (TLPE) successfully resulted in the simultaneous separation of diosgenin, untransformed steroidal saponins and glucose.     

Application of microwave‐assisted extraction coupled with high‐speed counter‐current chromatography for separation and purification of Dehydrocavidine from Corydalis saxicola Bunting

Introduction – Dehydrocavidine is a major component of Corydalis saxicola Bunting with sedative, analgesic, anticonvulsive and antibacterial activities. Conventional methods have disadvantages in extracting, separating and purifying dehydrocavidine from C. saxicola. Hence, an efficient method should be established. Objective – To develop a suitable preparative method in order to isolate dehydrocavidine from a complex C. saxicola extract by preparative HSCCC. Methodology – The methanol extract of C. saxicola was prepared by optimised microwave‐assisted extraction (MAE). The analytical HSCCC was used for the exploration of suitable solvent systems and the preparative HSCCC was used for larger scale separation and purification. Dehydrocavidine was analysed by high‐performance liquid chromatography (HPLC) and further identified by ESI‐MS and 1H NMR. Results – The optimised MAE experimental conditions were as follows: extraction temperature, 60°C; ratio of liquid to solid, 20; extraction time, 15 min; and microwave power, 700 W. In less than 4 h, 42.1 mg of dehydrocavidine (98.9% purity) was obtained from 900 mg crude extract in a one‐step separation, using a two‐phase solvent system composed of chloroform–methanol–0.3 m hydrochloric acid (4 : 0.5 : 2, v/v/v). Conclusion – Microwave‐assisted extraction coupled with high‐speed counter‐current chromatography is a powerful tool for extraction, separation and purification of dehydrocavidine from C. saxicola. Copyright © 2009 John Wiley & Sons, Ltd.     

Application of ion-pair high-performance liquid chromatography to detection of the atypical coproporphyrin isomers II and IV in human faeces

A highly selective and sensitive method has been developed for the detection of small amounts of the atypical isomers II and IV of coproporphyrin in human faeces. This method combines liquid—liquid extraction and solid-phase sampling techniques using talc and C₁₈-modified silica gel as the sorbents. Simultaneous separation of the four coproporphyrin isomers I–IV was achieved by isocratic ion-pair high-performance liquid chromatography. Stool samples of healthy subjects (n = 12) contained 1.1 ± 0.4% (mean ± S.D.) isomer II and 2.2 ± 0.9% isomer IV of total coproporphyrins. A somewhat higher content of isomer II (2.7%) and isomer IV (5.4%) was found in faeces of a patient suffering from porphyria variegata.     

Simultaneous analysis of diosgenin and sarsasapogenin in Asparagus officinalis byproduct by thin‐layer chromatography

Introduction – Asparagus officinalis L. has several biological activities including antifungal, antiviral and antitumoral activities due to the steroidal saponins. Normally diosgenin and sarsasapogenin are analysed separately by thin‐layer chromatography or high‐performance liquid chromatography (HPLC‐UV or HPLC‐ELSD), which is time‐consuming and expensive, so we need to find a rapid solution to this problem. Objective – To develop a sensitive, rapid and validated TLC method for simultaneous detection and quantification of diosgenin and sarsasapogenin. Methodology – Samples were prepared by extraction of A. officinalis with 70% aqueous ethanol to get steroidal saponins, and then hydrolysed using 36 mL 2 m hydrochloric acid for 3 h. The hydrolysis product was extracted with chloroform, and then analysed by TLC, the results of which were verified by HPLC and HPLC‐MS. Results – The retention factor (R_f) of diosgenin and sarsasapogenin on TLC plate were 0.49 and 0.6, respectively. After calculation from the regression equation of the standard curve, the contents of diosgenin and sarsasapogenin in the A. officinalis extract were 0.27–0.46 and 0.11–0.32%, respectively. Conclusion – The study showed that thin‐layer chromatography can be applied for the determination of diosgenin and sarsasapogenin in the oldest tissue of A. officinalis, and also can be conducted for screening of sapogenin in other plant or extracts. Copyright © 2010 John Wiley & Sons, Ltd.     

Comparison of plasma sample purification by manual liquid–liquid extraction, automated 96-well liquid–liquid extraction and automated 96-well solid-phase extraction for analysis by high-performance liquid chromatography with tandem mass spectrometry

Three extraction procedures were developed for the quantitative determination of a carboxylic acid containing analyte (I) in human plasma by high-performance liquid chromatography (HPLC) with negative ion electrospray tandem mass spectrometry (MS–MS). The first procedure was based on the manual liquid–liquid extraction (LLE) of the acidified plasma samples with methyl tert.-butyl ether. The second procedure was based on the automation of the manual LLE procedure using 96-well collection plates and a robotic liquid handling system. The third approach was based on automated solid-phase extraction (SPE) using 96-well SPE plates and a robotic liquid handling system. A lower limit of quantitation of 50 pg/ml was achieved using all three extraction procedures. The total time required to prepare calibration curve standards, aliquot the standards and plasma samples, and process a total of 96 standards and samples by manual LLE was three-times longer than the time required for 96-well SPE or 96-well LLE (4 h, 50 min vs. 1 h, 43 min). Even more importantly, the time the bioanalyst physically spent on the 96-well LLE or 96-well SPE procedure was only a small fraction of the time spent on the manual LLE procedure (<10 min vs. 4 h, 10 min). It should be noted that the 96-well SPE procedure incorporated the two steps of evaporation of the eluates to dryness and subsequent reconstitution of the dried extract. The total time required for the 96-well SPE could be reduced by 50% if the eluates were injected directly, eliminating the drying and reconstitution steps, which is achievable when sensitivity is less of an issue.