Quantitation of 5-Methyltetrahydrofolic Acid in Dried Blood Spots and Dried Plasma Spots by Stable Isotope Dilution Assays

Because of minimal data available on folate analysis in dried matrix spots (DMSs), we combined the advantages of stable isotope dilution assays followed by LC-MS/MS analysis with DMS sampling to develop a reliable method for the quantitation of plasma 5-methyltetrahydrofolic acid in dried blood spots (DBSs) and dried plasma spots (DPSs) as well as for the quantitation of whole blood 5-methyltetrahydrofolic acid in DBSs. We focused on two diagnostically conclusive parameters exhibited by the plasma and whole blood 5-methyltetrahydrofolic acid levels that reflect both temporary and long-term folate status. The method is performed using the [²H₄]-labeled isotopologue of the vitamin as the internal standard, and three steps are required for the extraction procedure. Elution of the punched out matrix spots was performed using stabilization buffer including Triton X-100 in a standardized ultrasonication treatment followed by enzymatic digestion (whole blood only) and solid-phase extraction with SAX cartridges. This method is sensitive enough to quantify 27 nmol/L whole blood 5-methyltetrahydrofolic acid in DBSs and 6.3 and 4.4 nmol/L plasma 5-methyltetrahydrofolic acid in DBSs and DPSs, respectively. The unprecedented accurate quantification of plasma 5-methyltetrahydrofolic acid in DBSs was achieved by thermal treatment prior to ultrasonication, inhibiting plasma conjugase activity. Mass screenings are more feasible and easier to facilitate for this method in terms of sample collection and storage compared with conventional clinical sampling for the assessment of folate status.     

Study of dried blood spots technique for the determination of dextromethorphan and its metabolite dextrorphan in human whole blood by LC–MS/MS

Dried blood spots (DBSs) technology was evaluated in an assay for the quantitation of dextromethorphan (DM) and its metabolite, dextrorphan (DT), in human whole blood using high performance liquid chromatography with tandem mass spectrometry method (LC–MS/MS). Both the parent drug and metabolite were spiked in the blood matrix and subsequently allowed to dry on a specimen collection card. The dried blood spots were removed using a manual punch and then extracted into methyl tert-butyl ether (MTBE). The organic supernatant was transferred and evaporated and the residue was reconstituted in 20% acetonitrile. The overall method recovery of DM and DT was 87.8% and 95.4%, respectively. The assay was linear over the concentration range of 0.2–200 ng/mL for both analytes. Several factors that potentially affect DBS assay quantitation were investigated, such as punch size, DBS sample punch-out location, and the volume of the blood sample pipetted on the specimen collection cards. The study determined that punch size does not affect assay quantitation accuracy. Indeed, a larger punch size increases the sensitivity due to the larger sampled blood spots. Sampling from different location on the specimen collection cards shows no significant variation for both drugs. The study also shows that acceptable results can be achieved with some variation of the sample volume, which allows a simple blood sampling procedure at the test sites. To achieve the similar lower limit of quantitation (LLOQ) as the plasma assay, several blood spots at the same concentration level were stacked together and extracted. Bioanalytical assays using the DBS technique are promising given the advantages of the method over the plasma assay.     

Application of dried blood spots combined with HPLC-MS/MS for the quantification of acetaminophen in toxicokinetic studies

A reversed phase HPLC-MS/MS method has been developed and validated for the quantitative bioanalysis of acetaminophen in dried blood spots (DBS) prepared from small volumes (15 microL) of dog blood. Samples were extracted for analysis with methanol. Detection was by positive ion TurboIonSpray ionisation combined with selected reaction monitoring MS. The analytical concentration range was 0.1-50 microg/mL. The intra-day precision and bias values were both less than 15%. Acetaminophen was stable in DBS stored at room temperature for at least 10 days. The methodology was applied in a toxicokinetic (TK) study where the data obtained from DBS samples was physiologically comparable with results from duplicate blood samples (diluted 1:1 (v/v) with water) analysed using identical HPLC-MS/MS conditions. This work demonstrates that quantitative analysis of a drug extracted from DBS can provide high quality TK data while minimising the volume of blood withdrawn from experimental animals, to an order of magnitude lower than is current practice in the pharmaceutical industry. This is the first reported application of DBS analysis to a TK study in support of a safety assessment study. The success of this and similar, related studies has led to the intent to apply DBS technology as the recommended analytical approach for the assessment of pharmacokinetics (PK)/TK for all new oral small molecule drug candidates, which have previously demonstrated a successful bioanalytical validation.     

Determination of moxifloxacin in dried blood spots using LC-MS/MS and the impact of the hematocrit and blood volume

Moxifloxacin (MFX) is a potential oral agent use in the treatment of multidrug-resistance tuberculosis (MDR-TB). Due to variability in pharmacokinetics and in vitro susceptibility of causative bacteria, therapeutic drug monitoring (TDM) of MFX is recommended. Conventional plasma sampling for TDM is facing logistical challenges, especially in limited resource areas, and dried blood spots (DBS) sampling may offer a chance to overcome this problem. The objective of this study was to develop a LC-MS/MS method for determination of MFX in dried blood spots (DBS) that is applicable for TDM. The influence of paper type, the hematocrit (Hct) and the blood volume per spot (V(b)) on the estimated blood volume in a disc (V(est)) was investigated. The extracts of 8mm diameter discs punched out from DBS were analyzed using liquid chromatography tandem mass spectrometry (LC-MS/MS) with cyanoimipramin as internal standard. The method was validated with respect to selectivity, linearity, accuracy, precision, sensitivity, recovery and stability. The effect of Hct and V(b) on LC-MS/MS analytical result was also investigated. The relationship between MFX concentrations in venous and finger prick DBS and those in plasma was clinically explored. V(est) was highly influenced by Hct while the effect of V(b) appeared to be different among paper types. Calibration curves were linear in the range of 0.05-6.00 mg/L with inter-day and intra-day precisions and biases of less than 11.1%. The recovery was 84.5, 85.1 and 92.6% in response to blood concentration of 0.15, 2.50 and 5.00 mg/L, respectively. A matrix effect of less than 11.9% was observed. MFX in DBS was stable for at least 4 weeks at room condition (temperature of 25°C and humidity of 50%). A large range of Hct value produced a significant analytical bias and it can be corrected with resulting DBS size. A good correlation between DBS and plasma concentrations was observed and comparable results between venous DBS and finger prick DBS was attained. This fully validated method is suitable for determination of MFX in dried blood spot and applicable for TDM.     

A dried blood spots technique based LC-MS/MS method for the analysis of posaconazole in human whole blood samples

A rugged and robust liquid chromatographic tandem mass spectrometric (LC-MS/MS) method utilizing dried blood spots (DBS) was developed and validated for the analysis of posaconazole in human whole blood. Posaconazole fortified blood samples were spotted (15 μL) onto Ahlstrom Alh-226 DBS cards and dried for at least 2h. Punched spots were then extracted by using a mixture of acetonitrile and water containing stable labeled internal standard (IS). Posaconazole and its IS were separated from endogenous matrix components on a Kinetex? C18 column under gradient conditions with a mobile phase A consisting of 0.1% formic acid and a mobile phase B consisting of 0.1% formic acid in acetonitrile/methanol (70/30, v/v). The analyte and IS were detected using a Sciex API 4000 triple quadrupole LC-MS/MS system equipped with a TurboIonSpray? source operated in the positive ion mode. The assay was linear over the concentration range of 5-5000 ng/mL. The inter-run accuracy and precision of the assay were -1.8% to 0.8% and 4.0% to 10.4%, respectively. Additional assessments unique to DBS were investigated including sample spot homogeneity, spot volume, and hematocrit. Blood spot homogeneity was maintained and accurate and precise quantitation results were obtained when using a blood spot volume of between 15 and 35 μL. Human blood samples with hematocrit values ranging between 25% and 41% gave acceptable quantitation results. The validation results indicate that the method is accurate, precise, sensitive, selective and reproducible.     

费约果外植体灭菌及愈伤组织的诱导

以费约果(Feijoa sallowiana Berg.)嫩叶和带芽茎段作为外植体,研究费约果愈伤组织诱导的最佳条件和方法.结果表明:(1)最佳基本培养基是MS;(2)二步灭菌法优于一步灭菌法;(3)嫩叶和带芽茎段愈伤组织诱导的最佳活性炭浓度分别为2.0 g L-1和3.0 g L-1;(4)最佳青霉素浓度分别为200 mg L-1和50 mg L-1;(5)光培养方式优于暗培养方式.(6)费约果嫩叶诱导愈伤组织的最佳培养基是MS 2.0 mgL-1 2,4-D 1.0 mg L-1 KT(或0.1 mg L-1NAA);(7)带芽茎段的最佳培养基是MS 1.0 mg L-16-BA 0.5 mg L-1NAA 1.0 mg L-1GA或MS 2.0 mg L-16-BA 0.1 mgL-1NAA 1.0 mgL-1GA.     

Biomonitoring of 37 trace elements in blood samples from inhabitants of northern Germany by ICP–MS

The trace elements Ag, As, Au, B, Ba, Be, Bi, Cd, Ce, Co, Cs, Cu, Ga, Hf, Hg, In, La, Mn, Mo, Ni, Pb, Pd, Rb, Rh, Ru, Sb, Se, Sn, Sr, Te, Th, Tl, U, V, W, Y and Zr were determined in 130 human blood samples from occupationally non-exposed volunteers living in the greater area of Bremen in northern Germany. The blood samples were collected in lithium heparin monovettes developed for trace metal determination and were analysed by inductively coupled plasma mass spectrometry (ICP–MS) with an octopole-based collision/reaction cell. For sample introduction into the ICP, the blood samples were diluted 1/10 (V/V) with a 0.1% Triton-X-100 and 0.5% (V/V) ammonia solution. The method validation of our developed routine method is described for all 37 elements and results about internal and external quality assurance are discussed. Information on exposure conditions of all human subjects were collected by questionnaire-based interviews, including smoking habits, seafood consumption and the type of dental alloys in the teeth. Mean values, geometric mean values, ranges and selected percentiles of all elemental concentrations in human blood are presented, which helps toxicologists and clinical chemists planning research about exposition to metals and health effects caused by exposition to metals.     

梭梭(Haloxylon ammodendron)组织培养和快繁技术

为了优化梭梭分化苗生根的培养基配方和培养条件,以剪去幼根的梭梭无菌苗茎、叶部分作为外植体,通过芽生芽途径获得了梭梭再生苗,重点对生根培养基和培养条件进行了系统的研究,根据试验结果得出结论:梭梭腋芽诱导培养基为MS+6-BA 2 mg.L-1,壮苗培养基为MS+6-BA 1.5 mg.L-1+GA3 1.0 mg.L-1,生根培养基为1/4MS+5%蔗糖+IBA0.2 mg.L-1+NAA 0.8 mg.L-1,生根率为74%。     

Assay method for the perfluorooctyl bromide (perflubron) in rat blood by gas chromatography–mass spectrometry

This paper describes a GC–MS method (SIM mode) for the analysis of perfluorooctyl bromide (perflubron, I) in rat blood. The chromatographic separation was performed by injection in the split mode using a CP-select 624 CB capillary column. Following destruction of the emulsion by addition of ethanol, the analytical procedure involves a liquid–liquid extraction with 1,1,2-trichlorotrifluoroethane. The bis(F-butyl)ethene (II) was used as internal standard. Observed retention times were 3.22 min for I and 2.32 min for II. Two calibration curves were used; linear detection responses were obtained for concentrations ranging from 0.009 to 0.9 mg/ml and from 0.9 to 13.5 mg/ml. The extraction efficiency averaged 50% for I and 93% for II. Precision ranged from 0.7 to 14%, and accuracy was between 91 and 109%. The limit of quantification was 9 μg/ml. The method validation results indicate that the performance characteristics of the method fulfilled the requirements for assay method for use in pharmacokinetic studies.     

High-performance liquid chromatographic assay of metabolites of thioguanine and mercaptopurine in capillary blood

The main metabolites of the cytotoxic drugs thioguanine (6TG) and mercaptopurine (6MP) can be measured conveniently in red blood cells (RBC). Isolation of RBC, however, is laborious and requires some milliliters of blood. This HPLC assay allows measurements of thiopurine metabolites in very small blood samples obtained from the finger-tip. The metabolites, derivatives of 6TG and methylmercaptopurine (6MeMP), were extracted and hydrolized with perchloric acid to liberate the corresponding base. 6MeMP is completely transformed under these conditions to 4-amino-5-(methylthio)carbonyl imidazole. The chromatographic separation of 6TG and this imidazole was performed in a single run under isocratic conditions within 10 min using a 70 mm column. The quantification limit was 0.5 nmol/ml for 6TG and 3 nmol/ml blood for 6MeMP. The accuracy was 83% for 6TG (CV=3%) over the concentration range of 0.5-20 nmol/ml blood and 102% (CV=4%) for 6MeMP over the range of 3-150 nmol/ml blood. The intra-assay CV ranged from 5.4 to 7.4% for 6TG and from 6.2 to 10.6% for 6MeMP. The inter-assay CV was 7.5 and 9.5% in a pooled blood sample. The levels in RBC in whole blood were nearly coincident with those obtained in separated RBC, isolation of RBC therefore is not necessary for these measurements, if the drugs are given per os in the day before blood sampling. The concentration of 6MeMP nucleotides is more dependent on the given 6MP dose than the concentration of 6TG nucleotides. Intraindividual variations were small at unchanged drug doses, interindividual metabolite concentrations were highly variable.     

云南玛咖种子、块根愈伤组织的诱导培养

通过对玛咖种子和块根两种外植体愈伤组织初导培养基和增生培养基的筛选及防褐化处理试验,结果表明玛咖种子或根初导培养基最优组合分别为3/4MS+3 mg.L-16-BA+0.3 mg.L-1NAA+25 g.L-1蔗糖、MS+0.3 mg.L-16-BA+0.3mg.L-1NAA+25 g.L-1蔗糖;其种子和小穗的增生培养基最优组合分别为MS+(0.3~0.5 mg.L-1)6-BA+0.3mg.L-1NAA、MS+0.3 mg.L-16-BA+0.3 mg.L-1NAA;在特定培养条件下,对褐变有一定程度的控制。该研究为玛咖的良种选育提供技术依据。     

Feasibility of an on-line restricted access material/liquid chromatography/tandem mass spectrometry method in the rapid and sensitive determination of organophosphorus triesters in human blood plasma

A rapid on-line solid phase extraction/liquid chromatography/tandem mass spectrometry (SPE/LC/MS/MS) method using restricted access material (RAM) was developed for the simultaneous determination of eight organophosphorus triesters in untreated human blood plasma. In a process involving column-switching techniques, the analytes were enriched on the RAM column, separated using a C-18 analytical column and detected with LC/MS. Tandem mass spectrometry was used to characterize and quantify the analytes. To elucidate the fragmentation pathway of a number of the analytes, MS3 experiments using an ion trap mass spectrometer were performed. The matrix effects associated with using APCI and ESI interfaces were investigated. The recoveries obtained were in the range 60-92% (R.S.D.<6%), with estimated detection limits between 0.2 and 1.8 ng/ml of plasma, and the total analysis time was 27 min.     

珍稀濒危植物香果树胚性细胞悬浮系的建立和植株再生

以香果树(Emmenopterys henryi)未成熟种子为试材, 探讨不同的接种量、基本培养基、糖浓度及植物生长调节物质等对体细胞胚胎生长的影响, 建立稳定的香果树胚性细胞悬浮培养与植株再生体系。结果表明: 悬浮培养条件下, 最适接种量为2%(鲜重); 较适合的基本培养基为MS; 蔗糖浓度为1%时容易使球形胚聚合体愈伤化, 浓度为3%和6%适合球形胚聚合体增殖, 浓度为9%则容易使球形胚聚合体褐化; 添加0.5 mg.L-16-BA 和0.5 mg.L-1 NAA的MS液体培养基, 当初始蔗糖浓度为3%, 然后逐步提高到6%则有利于香果树各个发育阶段的同步化; 子叶胚转到不含任何植物生长调节物质的MS固体培养基中可以长成正常植株。     

Determination of baicalin in rat cerebrospinal fluid and blood using microdialysis coupled with ultra-performance liquid chromatography-tandem mass spectrometry

An in vivo microdialysis sampling method coupled with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was employed for continuous simultaneous monitoring of unbound baicalin in rat blood and brain. Microdialysis probes were inserted into the jugular vein and brain cerebrospinal fluid (CSF) of Sprague-Dawley rats then, following administration of baicalin at doses of 24mg/kg via the candal vein, samples were collected every 20min and injected directly into the UPLC-MS/MS system. In vitro recoveries of the probes were 19.26% and 18.38%, while in vivo recoveries of the probes were 15.0% and 17.52% for blood and brain, respectively. This improved method offers a rapid quantitative procedure for the determination of baicalin with a retention time of only 1.6min. The lower limit of quantification (LLOQ) and the lower limit of detection (LLOD) based on a signal-to-noise ratio of 5 were 2.37 and 0.1ng/ml for anticoagulant citrate dextrose (ACD) solution, and 1.185 and 0.3ng/ml for artificial cerebrospinal fluid (aCSF), respectively. The pharmacokinetics results indicated that baicalin could pass through the blood-brain barrier (BBB) and was detectable in brain dialysate. These in vivo microdialysis-based measurements provide a technique for simple sampling and rapid sensitive analysis of unbound baicalin in rat blood and CSF and for further application in pharmacokinetic studies.     

Quantitative analysis of heterocyclic amines in urine by liquid chromatography coupled with tandem mass spectrometry

A sensitive, reproducible, and rapid analytical method for the analysis of trace-level heterocyclic amines (HCAs) that are expected to have high levels of human exposure was developed. Liquid–liquid extraction (LLE) with dichloromethane (DCM) followed by solid-phase extraction (SPE) was carried out. Liquid extraction with DCM under basic conditions was efficient in extracting HCAs from urine samples. For further purification, mixed mode cationic exchange (MCX) cartridges were applied to eliminate the remaining interferences after liquid extraction. Separation and quantification were performed by liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) in selected reaction monitoring (SRM) mode. The overall recoveries ranged between 71.0% and 113.6% with relative standard deviations (RSDs) of 5.1% to 14.7% for the entire procedure. The limits of detection (LODs) and limits of quantification (LOQs) of the proposed analytical method were in the ranges of 0.04 to 0.10 ng/ml and 0.15 to 0.36 ng/ml, respectively. This method was applied to the analysis of monitoring in urine samples for Korean school children, and the results demonstrated that the method can be used for the trace determination of HCAs in urine samples.     

Development of an LC‐MS method for simultaneous quantitation of amentoflavone and biapigenin,the minor and major biflavones from Hypericum perforatum L., in human plasma and its application to real blood

Introduction – Biflavones of Hypericum perforatum L. are bioactive compounds used in the treatment of inflammation and depression. Determination of amentoflavone and biapigenin from blood is challenging owing to their similar structures and low concentrations. Objective – To develop a rapid, sensitive and accurate method based on liquid‐phase extraction followed by high‐performance liquid chromatography and electrospray ionisation mass spectrometry (HPLC‐ESI‐MS) for quantification of biflavones in human plasma. Methodology – After extraction from blood, the analytes were subjected to HPLC with an XTerra® MS C₁₈ column and a binary mobile phase consisting of 2% formic acid in water and acetonitrile under isocratic elution conditions, with ESI‐MS detection in the negative ion mode and multiple reaction monitoring (MRM). Results – Both calibration curves showed good linearity within the concentration range 1–500 ng/mL. Limits of detection (S/N = 3) were 0.1 ng for pure substances and the limits of quantitation (S/N = 5) were 1.0 ng/mL from analyte‐spiked serum. The grand mean recovery was 90% from several subsamples of each biflavone. The imprecision (RSD) of peak areas was between 5% (intraday) and 10% (interday) for high concentrations (250 ng/mL) and between 10% (intraday) and 15% (interday) for low concentrations (1 ng/mL). Inaccuracy of the mean was less than 20% at the lower limit of quantitation. Conclusion – The developed and validated method for determination of biflavones from human plasma was effectively applied to pharmacokinetic studies of 13 probands and preliminary results indicate biphasic concentration–time curves. Copyright © 2010 John Wiley & Sons, Ltd.     

Analysis of glucosinolates, isothiocyanates, and amine degradation products in vegetable extracts and blood plasma by LC-MS/MS

Dietary glucosinolates are under intensive investigation as precursors of cancer-preventive isothiocyanates. Quantitation of the dose and bioavailability of glucosinolates and isothiocyanates requires a comprehensive analysis of the major dietary glucosinolates, isothiocyanates, and related metabolites. We report a liquid chromatography with tandem mass spectrometric detection (LC-MS/MS) analytical method for the comprehensive analysis of the seven major dietary glucosinolates, related isothiocyanates, and putative amine degradation products. The parent glucosinolates were sinigrin, gluconapin, progoitrin, glucoiberin, glucoraphanin, glucoalyssin, and gluconasturtiin. The LC-MS/MS analysis method for these compounds was developed and validated; a standard addition analysis protocol was used generally to avoid the requirement for stable isotopic standards. Where stable isotopic standards were available, internal standardization with these gave estimates in agreement with those obtained by the standard addition analysis protocol. For glucosinolates, negative ion electrospray LC-MS/MS analysis was performed. Isothiocyanates and amines were prederivatized to the corresponding thiourea and N-acetamides, respectively, and were quantified by positive ion electrospray LC-MS/MS. The limits of detection were 0.5-2 pmol; the recoveries for glucosinolates, isothiocyanates, and amines were 85-90%, 50-85%, and 60-70%, respectively; and the intra- and interbatch coefficients of variation were 1-4% and 3-10%, respectively. These methods provide facile access to comprehensive analytical data on the major dietary glucosinolates and related metabolites to quantify inputs and metabolic formation of these compounds in cancer prevention and related studies.     

Separation of Organoarsenicals by Means of Zwitterionic Hydrophilic Interaction Chromatography (ZIC®‐HILIC) and Parallel ICP‐MS/ESI‐MS Detection

An analytical scheme was developed for the separation and detection of organoarsenicals using a zwitterionic stationary phase of hydrophilic interaction chromatography (ZIC^®‐HILIC) coupled in parallel to electrospray ionization mass spectrometry (ESI‐MS) and to inductively coupled plasma mass spectroscopy (ICP‐MS). The optimization of separation and detection for organoarsenicals was mainly focused on the influence of the percentage of acetonitrile (MeCN) used as a major component of the mobile phase. Isocratic and gradient elution was applied by varying the MeCN percentage from 78 % to 70 % MeCN and 22 % to 30 % of an aqueous solution of ammonium acetate (125 mM NH₄Ac; pH 8.3) on a ZIC^®‐HILIC column (150 × 2.1 mm id, 3.5 μm), to allow for the separation and successful detection of nine organoarsenicals (i.e., 3‐nitro‐4‐hydroxyphenylarsonic acid (roxarsone, Rox), phenylarsonic acid (PAA), p‐arsanilic acid (p‐ASA), phenylarsine oxide (PAO), dimethylarsinate (DMA), methylarsonate (MMA), arsenobetaine (AsB), arsenocholine (AsC) and trimethylarsine oxide (TMAO)) within 45 min. All analytes were prepared in the mobile phase. The flow rate of the mobile phase, the splitting ratio between ICP‐MS and ESI‐MS detection, and the oxygen addition were adapted to ensure that there appeared a stably burning inductively coupled plasma. Furthermore, the analytical method was evaluated by the identification and quantification of AsB in the reference material DORM‐2 (dogfish muscle) resulting in a 95‐% recovery with respect to the AsB concentration in the extract.     

Second-tier test for quantification of underivatized amino acids in dry blood spot for metabolic diseases in newborn screening

The quantitative analysis of amino acids (AAs) in single dry blood spot (DBS) samples is an important issue for metabolic diseases as a second-tier test in newborn screening. An analytical method for quantifying underivatized AAs in DBS was developed by using liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS). The sample preparation in this method is simple and ion-pairing agent is not used in the mobile phase that could avoid ion suppression, which happens in mass spectrometry and avoids damage to the column. Through chromatographic separation, some isomeric compounds could be identified and quantified, which cannot be solved through only appropriate multiple reactions monitoring transitions by MS/MS. The concentrations of the different AAs were determined using non-deuterated internal standard. All calibration curves showed excellent linearity within test ranges. For most of the amino acids the accuracy of extraction recovery was between 85.3 and 115 %, and the precision of relative standard deviation was <7.0 %. The 35 AAs could be identified in DBS specimens by the developed LC–MS/MS method in 17–19 min, and eventually 24 AAs in DBS were quantified. The results of the present study prove that this method as a second-tier test in newborn screening for metabolic diseases could be performed by the quantification of free AAs in DBS using the LC–MS/MS method. The assay has advantages of high sensitive, specific, and inexpensive merits because non-deuterated internal standard and acetic acid instead of ion-pairing agent in mobile phase are used in this protocol.     

Study of the Se-containing metabolomes in Se-rich yeast by size-exclusion-cation-exchange HPLC with the parallel ICP MS and electrospray orbital ion trap detection

Strong cation exchange HPLC with the parallel ICP MS and electrospray hybrid linear ion trap quadrupole orbital trap mass spectrometry (ESI Orbitrap MS) detection was developed for the study of the metabolomic pattern of selenium in selenium-rich yeast. The mobile phase composition (gradient of ammonium formate in 20% methanol) was optimized to obtain separation in conditions guaranteeing the identical ICP MS sensitivity during the entire chromatographic run and the compatibility with electrospray ionization. Twenty seven Se-containing metabolites observed in the HPLC-ICP MS chromatogram were identified by ESI Orbitrap MS based on the Se isotopic pattern, the accurate molecular mass, and the multistage fragmentation patterns. The method allowed for the first time the correlation of the differences observed in HPLC-ICP MS chromatography of water extracts of Se-rich yeast samples from different manufacturers with the identity of the eluted compounds determined by ESI MS.