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.     

LC-MS/MS检测癫痫患者神经递质类氨基酸

目的：建立液相色谱串联质谱同位素内标法检测神经递质类氨基酸并用于癫痫患者临床评价。方法：选用AAA-C18柱色谱柱,以乙腈水（含有0.01%七氟丁酸、0.1%甲酸）为流动相,采用梯度洗脱进行分离,血浆样品用iTRAQ-115衍生化试剂处理后,加入iTRAQ-114衍生化的氨基酸内标并进样,选用3200QTRAP型质谱仪的多重反应监测（MRM）扫描方式进行检测。疾病组与健康组的统计采用t检验和主成份分析。结果：疾病组和健康组氨基酸测定结果显示：Trp、GABA两组间没有显著性差异（P〉0.05）,Arg、Gly、Ser、Tau、Asp、Glu、EtN、两组间有显著性差异（P〈0.05）,通过PCA分析显示,疾病组与健康组之间差异明显,Asp、Glu、Ser等是引起差异的主要氨基酸。结论：试验方法灵敏、专属性强,并初步的用于癫痫患者体内氨基酸评价。     

LC-MS/MS 检测癫痫患者神经递质类氨基酸

目的:建立液相色谱串联质谱同位素内标法检测神经递质类氨基酸并用于癫痫患者临床评价.方法:选用AAA-C18柱色谱柱,以乙腈水(含有0.01%七氟丁酸、0.1%甲酸)为流动相,采用梯度洗脱进行分离,血浆样品用iTRAQ-115衍生化试剂处理后,加入iTRAQ-114衍生化的氨基酸内标并进样,选用3200QTRAP型质谱仪的多重反应监测(MRM)扫描方式进行检测.疾病组与健康组的统计采用t检验和主成份分析.结果:疾病组和健康组氨基酸测定结果显示:Trp、GABA两组间没有显著性差异(P＞0.05),Arg、Glv、Ser、Tau、Asp、Glu、EtN、两组间有显著性差异(P＜0.05),通过PCA分析显示,疾病组与健康组之间差异明显,Asp、Glu、Ser等是引起差异的主要氨基酸.结论:试验方法灵敏、专属性强,并初步的用于癫痫患者体内氨基酸评价.     

Quantification of urinary zwitterionic organic acids using weak-anion exchange chromatography with tandem MS detection

A rapid and accurate quantitative method was developed and validated for the analysis of four urinary organic acids with nitrogen containing functional groups, formiminoglutamic acid (FIGLU), pyroglutamic acid (PYRGLU), 5-hydroxyindoleacetic acid (5-HIAA), and 2-methylhippuric acid (2-METHIP) by liquid chromatography tandem mass spectrometry (LC/MS/MS). The chromatography was developed using a weak anion-exchange amino column that provided mixed-mode retention of the analytes. The elution gradient relied on changes in mobile phase pH over a concave gradient, without the use of counter-ions or concentrated salt buffers. A simple sample preparation was used, only requiring the dilution of urine prior to instrumental analysis. The method was validated based on linearity (r2>or=0.995), accuracy (85-115%), precision (C.V.<12%), sample preparation stability (相似文献   

Development and validation of a dried blood spot—LC–APCI–MS assay for estimation of canrenone in paediatric samples

A selective and sensitive liquid chromatography (LC)–atmospheric pressure chemical ionisation (APCI)–mass spectroscopic (MS) assay of canrenone has been developed and validated employing Dried Blood Spots (DBS) as the sample collection medium. DBS samples were prepared by applying 30 μl of spiked whole blood onto Guthrie cards. A 6 mm disc was punched from the each DBS and extracted with 2 ml of methanolic solution of 17α-methyltestosterone (Internal Standard). The methanolic extract was evaporated to dryness and reconstituted in acetonitrile:water (1:9, v/v). The reconstituted solution was further subjected to solid phase extraction using HLB cartridges. Chromatographic separation was achieved using Waters Sunfire C18 reversed-phase column using isocratic elution, followed by a high organic wash to clear late eluting/highly retained components. The mobile phase consisted of methanol:water (60:40, v/v) pumped at a flow rate of 0.3 ml/min. LC–APCI–MS detection was performed in the selected-ion monitoring (SIM) mode using target ions at m/z 341.1 and 303.3 for canrenone and internal standard respectively. The selectivity of the method was established by analysing DBS samples from 6 different sources (individuals). The calibration curve for canrenone was found to be linear over 25–1000 ng/ml (r > 0.994). Accuracy (% RE) and precision (% CV) values for within and between day were <20% at the lower limit of quantification (LLQC) and <15% at all other concentrations tested. The LLOQ of the method was validated at 25 ng/ml. Clinical validation of the method was achieved by employing the validated method for analysis of 160 DBS samples from 37 neonatal and paediatric patients.     

Application of the Phenomenex EZ:faast™ amino acid analysis kit for rapid gas-chromatographic determination of concentrations of plasma tryptophan and its brain uptake competitors

The Phenomenex EZ:faast™ amino acid analysis kit is available for gas (GC) or liquid (LC) chromatographic analysis of amino acids (AA) using mass spectrometry (MS) and other GC detectors. We used it for rapid GC determination of plasma tryptophan, its brain uptake competitors (Val, Leu, Ile, Phe and Tyr) and many other amino acids. Based on solid-phase extraction, this fast method enables one person to process two plasma samples in 8–10 min and six samples in ∼15 min up to GC injection and a 7-min GC run per plasma sample. Using a Perkin-Elmer Clarus 500 GC, a Total Chrome software, a flame-ionisation detector (FID) and norvaline as internal standard, we used this method to analyse ∼1,000 plasma samples from normal subjects undergoing acute tryptophan depletion and loading tests. The limit of detection for most amino acids is 1 nmol/ml (1 μM) and in many cases less. With manual injection, coefficients of variation for the above six amino acids were 1.5–6.2% (intra-assay) and 3.8–9.7% (inter-assay). This simple, rapid and elegant method will be valuable to the amino acid analyst and researcher, as it can save much manpower time and meet urgent emergency requests and the demands of a high-throughput laboratory.     

Modifications in electrospray tandem mass spectrometry for a neonatal-screening pilot study in Japan

In a neonatal-screening pilot study for inherited disorders in organic acid and amino acid metabolism, we analyzed butyrated acylcarnitines and amino acids in blood spots of more than 20 000 newborns by electrospray tandem mass spectrometry. In order to screen urea cycle disorders, we performed multiple scanning functions with additional stable isotope-labelled internal standards, since such reported functions as neutral loss of m/z 102 or 109 for butyrated amino acids were not sufficient. Arginine levels were measured with arginine-¹³C₆. Hypocitrullinemia for the screening of some urea cycle disorders was detectable by measurement with synthesized citrulline-d₆, although we did not find any such disorders. In the acylcarnitine analysis, we found a patient with propionic acidemia, who has been treated effectively. The increasing false positive rate due to the use of pivalic acid-containing antibiotics in the diagnosis of isovaleric acidemia was a problem in Japan.     

Sensitive assay for determining plasma tenofovir concentrations by LC/MS/MS

An LC/MS/MS assay for the determination of tenofovir (TNF) was developed and validated for use with the EDTA anticoagulated human plasma matrix. Heparin-treated plasma and serum matrices were also validated. After addition of adefovir as an internal standard, trifluoroacetic acid was used to produce a protein-free extract. Chromatographic separation was achieved with a Polar-RP Synergi, 2.0 mm x 150 mm, reversed-phase analytical column. The mobile phase was 3% acetonitrile/1% acetic acid, aq. Detection of TNF and the internal standard was achieved by ESI MS/MS in the positive ion mode using 288/176 and 274/162 transitions, respectively. The method was linear from 10 to 750 ng/ml with a minimum quantifiable limit of 10 ng/ml when 250 microl aliquots were analyzed. The usefulness of this LC/MS/MS method to routinely monitor plasma concentrations of TNF was demonstrated along with its ability to assist in the performance of pharmacokinetic studies.     

Urinary amino acid analysis: A comparison of iTRAQ®–LC–MS/MS,GC–MS,and amino acid analyzer

Urinary amino acid analysis is typically done by cation-exchange chromatography followed by post-column derivatization with ninhydrin and UV detection. This method lacks throughput and specificity. Two recently introduced stable isotope ratio mass spectrometric methods promise to overcome those shortcomings. Using two blinded sets of urine replicates and a certified amino acid standard, we compared the precision and accuracy of gas chromatography/mass spectrometry (GC–MS) and liquid chromatography–tandem mass spectrometry (LC–MS/MS) of propyl chloroformate and iTRAQ^® derivatized amino acids, respectively, to conventional amino acid analysis. The GC–MS method builds on the direct derivatization of amino acids in diluted urine with propyl chloroformate, GC separation and mass spectrometric quantification of derivatives using stable isotope labeled standards. The LC–MS/MS method requires prior urinary protein precipitation followed by labeling of urinary and standard amino acids with iTRAQ^® tags containing different cleavable reporter ions distinguishable by MS/MS fragmentation. Means and standard deviations of percent technical error (%TE) computed for 20 amino acids determined by amino acid analyzer, GC–MS, and iTRAQ^®–LC–MS/MS analyses of 33 duplicate and triplicate urine specimens were 7.27 ± 5.22, 21.18 ± 10.94, and 18.34 ± 14.67, respectively. Corresponding values for 13 amino acids determined in a second batch of 144 urine specimens measured in duplicate or triplicate were 8.39 ± 5.35, 6.23 ± 3.84, and 35.37 ± 29.42. Both GC–MS and iTRAQ^®–LC–MS/MS are suited for high-throughput amino acid analysis, with the former offering at present higher reproducibility and completely automated sample pretreatment, while the latter covers more amino acids and related amines.     

Rapid and Precise Measurement of Serum Branched-Chain and Aromatic Amino Acids by Isotope Dilution Liquid Chromatography Tandem Mass Spectrometry

Background

Serum branched-chain and aromatic amino acids (BCAAs and AAAs) have emerged as predictors for the future development of diabetes and may aid in diabetes risk assessment. However, the current methods for the analysis of such amino acids in biological samples are time consuming.

Methods

An isotope dilution liquid chromatography tandem mass spectrometry (ID-LC/MS/MS) method for serum BCAAs and AAAs was developed. The serum was mixed with isotope-labeled BCAA and AAA internal standards and the amino acids were extracted with acetonitrile, followed by analysis using LC/MS/MS. The LC separation was performed on a reversed-phase C₁₈ column, and the MS/MS detection was performed via the positive electronic spray ionization in multiple reaction monitoring mode.

Results

Specific analysis of the amino acids was achieved within 2 min. Intra-run and total CVs for the amino acids were less than 2% and 4%, respectively, and the analytical recoveries ranged from 99.6 to 103.6%.

Conclusion

A rapid and precise method for the measurement of serum BCAAs and AAAs was developed and may serve as a quick tool for screening serum BCAAs and AAAs in studies assessing diabetes risk.     

Minimally permutated peptide analogs as internal standards for relative and absolute quantification of peptides and proteins

A novel type of isobaric internal peptide standard for quantitative proteomics is described. The standard is a synthetic peptide derived from the target peptide by positional permutation of two amino acids. This type of internal standard is denominated minimally permutated peptide analog (MIPA). MIPA can be differentiated from their target analytes by LC‐MS due to individual retention times and/or by MS/MS due to specific fragment ions. Both quantification methods are demonstrated using peptide mixtures of low and high complexity.     

A newborn screening method for cerebrotendinous xanthomatosis using bile alcohol glucuronides and metabolite ratios

Cerebrotendinous xanthomatosis (CTX) is a treatable neurodegenerative metabolic disorder of bile acid synthesis in which symptoms can be prevented if treatment with chenodeoxycholic acid supplementation is initiated early in life, making CTX an excellent candidate for newborn screening. We developed a new dried blood spot (DBS) screening assay for this disorder on the basis of different ratios between the accumulating cholestanetetrol glucuronide (tetrol) and specific bile acids/bile acid intermediates, without the need for derivatization. A quarter-inch DBS punch was extracted with methanol, internal standards were added, and after concentration the extract was injected into the tandem mass spectrometer using a 2 min flow injection analysis for which specific transitions were measured for cholestanetetrol glucuronide, taurochenodeoxycholic acid (t-CDCA), and taurotrihydroxycholestanoic acid (t-THCA). A proof-of-principle experiment was performed using 217 Guthrie cards from healthy term/preterm newborns, CTX patients, and Zellweger patients. Using two calculated biomarkers, tetrol:t-CDCA and t-THCA:tetrol, this straightforward method achieved an excellent separation between DBSs of CTX patients and those of controls, Zellweger patients, and newborns with cholestasis. The results of this small pilot study indicate that the tetrol:t-CDCA ratio is an excellent derived biomarker for CTX that has the potential to be used in neonatal screening programs.     

Clinical assay of four thiol amino acid redox couples by LC–MS/MS: Utility in thalassemia

The total concentrations of four sulfur amino acid (SAA) metabolite redox couples (reduced and oxidized forms of homocysteine, cysteine, glutathione, and cysteinylglycine) in human blood are assayed with a simple and sensitive method by liquid chromatography–electrospray positive ionization–tandem mass spectrometry. To prevent ex vivo thiol oxidation, iodoacetamide (IAM) is used immediately following the blood draw. To selectively enrich for S-carboxyamidomethylated SAA, and other cationic amino acids metabolites, proprietary strong cation-exchange solid phase extraction tips are used. Analytes are further derivatized with isopropylchloroformate (IPCF) to esterify the amino and the carboxylic groups. Double derivatization with IAM and IPCF improves the reverse phase liquid chromatography separation of SAA metabolites. The use of detection mode of multiple-reaction monitoring (MRM) allows sensitive and specific simultaneous detection of SAA. The internal standards used to account for the matrix effects of human plasma and erythrocytes were plant glutathione analogue, homoglutathione, and stable isotopes of cystine and homocystine. The method was validated for its linearity, accuracy, and precision. Excellent linearity of detection (r² > 0.98) was observed over relevant ranges for plasma and erythrocyte samples, and the limits of detection were established to be between 5 and 20 nM. Relative standard deviations were <9% for within-day variations and <15% for between-day variations. The method was used to assess thiol redox states in plasma and erythrocytes isolated from healthy subjects and thalassemia patients.     

Reliable and sensitive analysis of amino acids in the peptidoglycan of actinomycetes using the advanced Marfey's method

For the chemotaxonomic classification of actinomycetes, we developed a new method for the detection of the 2, 6-diaminopimelic acid (A(2)pm) stereoisomers and 3-hydroxy diaminopimelic acid (3-OH A(2)pm) in the cell wall peptidoglycan of actinomycetes using "the advanced Marfey's method", which consists of a chromatographic technique for the separation of amino acids into each of its enantiomers by derivatization with 1-fluoro-2, 4-dinitrophenyl-5-L-luecinamide (L-FDLA) and D-FDLA, and a detection method using liquid chromatography/mass spectrometry (LC/MS). This method was successfully applied to determine the absolute configuration of the A(2)pm and detect the 3-OH A(2)pm included in the cell wall peptidoglycan of the standard strains. Because the procedure can be performed with very small amounts of an amino acid, it was possible to use one colony from an agar plate of the actinomycetes for the acid hydrolysis. In addition, the constituent amino acids and their absolute configurations in the cell wall of the actinomycetes could be simultaneously determined. Thus, a reliable, sensitive and rapid analytical method for amino acids including A(2)pm in the peptidoglycan of the microorganisms was established.     

Simultaneous analysis of the non-canonical amino acids norleucine and norvaline in biopharmaceutical-related fermentation processes by a new ultra-high performance liquid chromatography approach

In this study, a precise and reliable ultra-high performance liquid chromatography (UHPLC) method for the simultaneous determination of non-canonical (norvaline and norleucine) and standard amino acids (aspartic acid, glutamic acid, serine, histidine, glycine, threonine, arginine, tyrosine, methionine, valine, phenylalanine, isoleucine, leucine) in biopharmaceutical-related fermentation processes was established. After pre-column derivatization with ortho-phthaldialdehyde and 2-mercaptoethanol, the derivatives were separated on a sub-2 μm particle C₁₈ reverse-phase column. Identification and quantification of amino acids were carried out by fluorescence detection. To test method feasibility on standard HPLC instruments, the assay was properly transferred to a core–shell particle C₁₈ reverse-phase column. The limits of detection showed excellent sensitivity by values from 0.06 to 0.17 pmol per injection and limits of quantification between 0.19 and 0.89 pmol. In the present study, the newly established UHPLC method was applied to a recombinant antibody Escherichia coli fermentation process for the analysis of total free amino acids. We were able to specifically detect and quantify the unfavorable amino acids in such complex samples. Since we observed trace amounts of norvaline and norleucine during all fermentation phases, an obligatory process monitoring should be considered to improve quality of recombinant protein drugs in future.     

Analysis of amino acids by HPLC/electrospray negative ion tandem mass spectrometry using 9-fluorenylmethoxycarbonyl chloride (Fmoc-Cl) derivatization

A new method for the determination of amino acids is presented. It combines established methods for the derivatization of primary and secondary amino groups with 9-fluorenylmethoxycarbonyl chloride (Fmoc-Cl) with the subsequent amino acid specific detection of the derivatives by LC–ESI–MS/MS using multiple reaction monitoring (MRM). The derivatization proceeds within 5 min, and the resulting amino acid derivatives can be rapidly purified from matrix by solid-phase extraction (SPE) on HR-X resin and separated by reversed-phase HPLC. The Fmoc derivatives yield several amino acid specific fragment ions which opened the possibility to select amino acid specific MRM transitions. The method was applied to all 20 proteinogenic amino acids, and the quantification was performed using l-norvaline as standard. A limit of detection as low as 1 fmol/µl with a linear range of up to 125 pmol/µl could be obtained. Intraday and interday precisions were lower than 10 % relative standard deviations for most of the amino acids. Quantification using l-norvaline as internal standard gave very similar results compared to the quantification using deuterated amino acid as internal standards. Using this protocol, it was possible to record the amino acid profiles of only a single root from Arabidopsis thaliana seedlings and to compare it with the amino acid profiles of 20 dissected root meristems (200 μm).     

Quantification of 22 plasma amino acids combining derivatization and ion-pair LC-MS/MS

Time efficient and comprehensive quantification of amino acids continues to be a challenge. We developed a sensitive and precise method for quantitative analysis of amino acids from very small plasma and serum volumes. Ion-pair chromatography of amino acid butyl esters proved to provide an optimal combination of selectivity, sensitivity and robustness. 10 μL of plasma or serum are added to precipitation reagent containing stable isotope standards. After protein precipitation, the supernatants is dried and incubated with 3N butanolic HCl for improving chromatographic separation and ionization efficiency. Amino acid butyl esters are separated using ion-pair (heptafluorobutyric acid) reversed-phase chromatography coupled to triple quadrupole mass spectrometry. The established method enables quantitative analysis of 22 amino acids, all 20 proteinogenic amino acids, ornithine and citrulline. Cysteine is measured as cystine. The combination of precipitation, derivatization and chromatographic separation effectively avoids ion suppression and coelution. Simultaneous with quantification, analyte identity is verified in each sample using qualifier ions. The micro-method is very sensitive and accurate. The intra-assay precision for the analysis of plasma was 2.6-10.1%. Absolute accuracy as determined by comparison of external reference samples was 82-117.7%. Excellent linearity of detection response was demonstrated for all compounds in the range representative for clinical samples from infants and adults. Lower limits of quantification were in the range of 1 μmol/L for all analytes. In conclusion, the method is ideally suited for cost-effective high-throughput analysis of large numbers of samples in clinical studies and metabolomics research.     

Quantitation of fourteen urinary alpha-amino acids using isobutane gas chromatography chemical ionization mass spectrometry with 13C amino acids as internal standards

Isobutane chemical ionization gas chromatography mass spectometry of the N-trifluoroacetyl-carboxy-n-butyl ester derivatives of amino acids, using a commercial per-13C-amino acid mixture as internal standards, provided a sensitive and specific method for quantitative analysis of fourteen urinary alpha-amino acids. A computer controlled quadrupole mass spectrometer was used in a selected ion monitoring mode to record the ion current due to the protonated molecular ions of each alpha-amino acid/13C analogue pair. BASIC programmes located peak maxima, and using previously established standard curves, calculated the amino acid content on the bases of both peak height and peak area ratios. Duplicate amino acid analyses are possible on 5 microliter of urine. Instrumental analysis required 25 minutes, automated data processing 10 minutes, and sample preparation 2 hours. Detection limits approached 1 ng with a typical mean standard deviation of 2% for the instrumental analysis.     

Liquid chromatography MALDI MS/MS for membrane proteome analysis

Membrane proteins play critical roles in many biological functions and are often the molecular targets for drug discovery. However, their analysis presents a special challenge largely due to their highly hydrophobic nature. We present a surfactant-aided shotgun proteomics approach for membrane proteome analysis. In this approach, membrane proteins were solubilized and digested in the presence of SDS followed by newly developed auto-offline liquid chromatography/matrix-assisted laser desorption ionization (LC/MALDI) tandem MS analysis. Because of high tolerance of MALDI to SDS, one-dimensional (1D) LC separation can be combined with MALDI for direct analysis of protein digests containing SDS, without the need for extensive sample cleanup. In addition, the heated droplet interface used in LC/MALDI can work with high flow LC separations, allowing a relatively large amount of protein digest to be used for 1D LC/MALDI which facilitates the detection of low abundance proteins. The proteome identification results obtained by LC/MALDI are compared to the gel electrophoresis/MS method as well as the shotgun proteomics method using 2D LC/electrospray ionization MS. It is demonstrated that, while LC/MALDI provides more extensive proteome coverage compared to the other two methods, these three methods are complementary to each other and a combination of these methods should provide a more comprehensive membrane proteome analysis.     

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.