New marker of bone resorption: hydroxyproline-containing peptide High-performance liquid chromatographic assay without hydrolysis as an alternative to hydroxyproline determination: a preliminary report

A high-performance liquid chromatographic (HPLC) assay for a urinary hydroxyproline-containing peptide (hydroxyproline peptide, HypP) is described. This peptide represents about 50% of urinary hydroxyproline-containing peptides. Its concentration and total 4-hydroxyproline (Hyp) concentration evaluated in 325 urine samples have been shown to be closely correlated (r = 0.972; y = 0.499x − 1.5), which may indicate that the two markers provide the same information. The HypP assay, similar to Hyp assay, is carried out without hydrolysis of urine samples. After the blocking of primary amino acids by o-phthaldialdehyde (OPA) and derivatization of secondary amino acids by 9-fluorenylmethyl chloroformate (FMOC-Cl), the FMOC derivatives of HypP and 3,4-dehydroproline (internal standard) were separated on a strong anion-exchange column and detected fluorimetrically. HypP concentration was calculated by measurement of peak-area ratios of HypP and the hydroxyproline standard. The HypP/creatinine (mmol/mol) ratio in fasting urine samples from healthy adults was found to be 8.2 (S.D. = 1.6, n = 33) in 27–44-year-old premenopausal women and 6.9 (S.D. = 1.7, n = 21) in 28–49-year-old men.     

A three-phase liquid chromatographic method for δC analysis of amino acids from biological protein hydrolysates using liquid chromatography–isotope ratio mass spectrometry

We report a three-phase chromatographic method for the separation and analysis of δ¹³C values of underivatized amino acids from biological proteins (keratin, collagen, and casein) using liquid chromatography–isotope ratio mass spectrometry (LC–IRMS). Both precision and accuracy of δ¹³C values for standard amino acid mixtures over the range of approximately 8 to 1320 ng of carbon per amino acid on the column were assessed. The precision of δ¹³C values of amino acids was found to be better at higher concentrations, whereas accuracy improved at lower concentrations. The optimal performance for this method was achieved with between 80 and 660 ng of carbon of each amino acid on the column. At amino acid amounts lower than 20 ng of carbon on the column, precision and accuracy may become compromised. The application of this new three-phase chromatographic technique will allow the analysis of δ¹³C of amino acids to be carried out as a routine method and benefit fields of research such as biomedicine, forensics, ecology, nutrition, and palaeodiet reconstruction in archaeology.     

Analysis of 13C labeling enrichment in microbial culture applying metabolic tracer experiments using gas chromatography-combustion-isotope ratio mass spectrometry

The applicability of gas chromatography–combustion–isotope ratio mass spectrometry (GC–C–IRMS) for the quantification of ¹³C enrichment of proteinogenic amino acids in metabolic tracer experiments was evaluated. Measurement of the ¹³C enrichment of proteinogenic amino acids from cell hydrolyzates of Corynebacterium glutamicum growing on different mixtures containing between 0.5 and 10% [1-¹³C]glucose shows the significance of kinetic isotope effects in metabolic flux studies at low degree of labeling. We developed a method to calculate the ¹³C enrichment. The approach to correct for these effects in metabolic flux studies using δ¹³C measurement by GC–C–IRMS uses two parallel experiments applying substrate with natural abundance and ¹³C-enriched tracer substrate, respectively. The fractional enrichment obtained in natural substrate is subtracted from that of the enriched one. Tracer studies with C. glutamicum resulted in a statistically identical relative fractional enrichment of ¹³C in proteinogenic amino acids over the whole range of applied concentrations of [1-¹³C]glucose. The current findings indicate a great potential of GC–C–IRMS for labeling quantification in ¹³C metabolic flux analysis with low labeling degree of tracer substrate directly in larger scale bioreactors.     

Validation of the determination of amino acids in plasma by high-performance liquid chromatography using automated pre-column derivatization with o-phthaldialdehyde

A sensitive and reproducible fully automated method for the determination of amino acids in plasma based on reversed-phase high-performance liquid chromatography and o-phthaldialdehyde pre-column derivatization is described. A 5-μm Spherisorb ODS 2 column (125 × 3 mm I.D.) was selected for routine determination. Over 40 physiological amino acids could be determined within 49 min (injection to injection) and 48 samples could be processed unattended. The coefficients of variation for most amino acids in plasma were below 4%. We were also able to measure trace amounts of amino acids in plasma normally not detected in a routine analysis. The results obtained with the method described compared favourably with those of conventional amino acid analysis (r = 0.997) and were in excellent agreement with those of other laboratories (r = 0.999).     

High‐performance liquid chromatography evaluation of the enantiomeric purity of amino acids by means of automated precolumn derivatization with ortho‐phthalaldehyde and chiral thiols

The use of ortho‐phthalaldehyde (OPA) for the derivatization of amino acids (AA) is well known. It enables the separation of the derivatives on common reversed phase columns and improves the sensitivity with fluorescence detection. With the use of a chiral thiol an indirect enantioseparation of chiral amines and AAs is feasible. The major drawback of the OPA‐derivatization is the poor stability of the products. Here, a method with an in‐needle derivatization procedure is optimized to facilitate a quantitative conversion of the AA with OPA and the chiral thiols N‐acetyl‐L‐cysteine or N‐isobutyryl‐L‐cysteine, followed by a subsequent analysis, eluding the stability issue. Both enantiomers of a single AA were separated as OPA‐derivatives with a pentafluorophenyl column and a gradient program consisting of 50 mM sodium acetate buffer pH = 5.0 and acetonitrile. Fluorescence detection is commonly used to achieve sufficient sensitivity. In this study, the enantiomeric impurity of an AA can be detected indirectly with common UV spectrophotometric detection with a limit of quantitation of 0.04%. Seventeen different L‐AAs were tested and the amount of D‐AA for each individual AA was calculated by means of area normalization, which ranged from not detectable up to 4.29%. The recovery of the minor enantiomer of L‐ and D‐AA was demonstrated for three AAs at a 0.04% level and ranged between 92.3 and 113.3%, with the relative standard deviation between 1.7 and 8.2%.     

Advancement in the derivatizations of the amino groups with the o-phthaldehyde-thiol and with the 9-fluorenylmethyloxycarbonyl chloride reagents

An overview is presented on the advancement of the two most frequently used derivatization protocols applying the o-phthalaldehyde (OPA)-thiol and the fluorenylmethyloxycarbonyl (FMOC) chloride reagents, prior to the high performance liquid chromatographic analysis of amino acids. This review pays special attention (i) to the blank value, to the composition, to the stability and to the life time of the reagents, (ii) to the optimum pH conditions for the interactions and derivatives' elutions, (iii) to the characteristics and behavior of those amino acids which might provide more than one derivative correlating with the molar ratios of the reagent to the reactants, and (iv) on the practical applicability of the optimized protocols.     

Optimization of amino acid type-specific <Superscript>13</Superscript>C and <Superscript>15</Superscript>N labeling for the backbone assignment of membrane proteins by solution- and solid-state NMR with the UPLABEL algorithm

We present a computational method for finding optimal labeling patterns for the backbone assignment of membrane proteins and other large proteins that cannot be assigned by conventional strategies. Following the approach of Kainosho and Tsuji (Biochemistry 21:6273–6279 (1982)), types of amino acids are labeled with ¹³C or/and ¹⁵N such that cross peaks between ¹³CO(i – 1) and ¹⁵NH(i) result only for pairs of sequentially adjacent amino acids of which the first is labeled with ¹³C and the second with ¹⁵N. In this way, unambiguous sequence-specific assignments can be obtained for unique pairs of amino acids that occur exactly once in the sequence of the protein. To be practical, it is crucial to limit the number of differently labeled protein samples that have to be prepared while obtaining an optimal extent of labeled unique amino acid pairs. Our computer algorithm UPLABEL for optimal unique pair labeling, implemented in the program CYANA and in a standalone program, and also available through a web portal, uses combinatorial optimization to find for a given amino acid sequence labeling patterns that maximize the number of unique pair assignments with a minimal number of differently labeled protein samples. Various auxiliary conditions, including labeled amino acid availability and price, previously known partial assignments, and sequence regions of particular interest can be taken into account when determining optimal amino acid type-specific labeling patterns. The method is illustrated for the assignment of the human G-protein coupled receptor bradykinin B2 (B₂R) and applied as a starting point for the backbone assignment of the membrane protein proteorhodopsin.     

Micellar electrokinetic chromatography of hydroxyproline and other secondary amino acids in biological samples with laser-induced fluorescence detection

Micellar electrokinetic chromatography (MEKC) with laser-induced fluorescence (LIF) was used for the rapid and sensitive detection of hydroxyproline in serum and hydrolyzed urine that were pre-column derivatized with 9-fluorenylmethyl chloroformate (FMOC). The application of the combined o-phthalaldehyde (OPA)/FMOC derivatization in MEKC for the selective detection of secondary amino acids in biological samples is investigated.     

Rapid measurement of plasma free fatty acid concentration and isotopic enrichment using LC/MS

Measurements of plasma free fatty acids (FFA) concentration and isotopic enrichment are commonly used to evaluate FFA metabolism. Until now, gas chromatography-combustion-isotope ratio mass spectrometry (GC/C/IRMS) was the best method to measure isotopic enrichment in the methyl derivatives of ¹³C-labeled fatty acids. Although IRMS is excellent for analyzing enrichment, it requires time-consuming derivatization steps and is not optimal for measuring FFA concentrations. We developed a new, rapid, and reliable method for simultaneous quantification of ¹³C-labeled fatty acids in plasma using high-performance liquid chromatography-mass spectrometry (HPLC/MS). This method involves a very quick Dole extraction procedure and direct injection of the samples on the HPLC system. After chromatographic separation, the samples are directed to the mass spectrometer for electrospray ionization (ESI) and analysis in the negative mode using single ion monitoring. By employing equipment with two columns connected parallel to a mass spectrometer, we can double the throughput to the mass spectrometer, reducing the analysis time per sample to 5 min. Palmitate flux measured using this approach agreed well with the GC/C/IRMS method. This HPLC/MS method provides accurate and precise measures of FFA concentration and enrichment.     

Reversed-phase high-performance liquid chromatographic analysis of hydroxyproline and proline from collagen by derivatization with dabsyl chloride

A high-performance liquid chromatographic method for the analysis of hydroxyproline and proline has been developed. The method is based on the derivatization of the secondary amino group with dabsyl-chloride after blocking of the primary amino group with o-phthalaldehyde. Dabsyl-hydroxyproline and dabsyl-proline were separated from other amino acids by high-performance liquid chromatography in the gradient elution mode, and eluted at 10.27 and 16.02 min, respectively. The correlations between the peak areas of dabsyl-hydroxyproline and dabsyl-proline were linear in the range from 20–200 pmol, with equations y = 1.10x − 0.80 (r = 0.999) and y = 1.12x − 0.52 (r = 0.999), respectively. The method was applied to the analysis of rat tail collagen, and the contents of hydroxyproline and proline were 1.55 ± 0.04 and 2.03 ± 0.04 nmol/μg, respectively.     

A fluorescent electrophilic reagent, 9-fluorenone-4-carbonyl chloride (FCC), for the enantioresolution of amino acids on a teicoplanin phase under the elution of the methanol-based solvent mixture

Summary. A fluorescent electrophilic reagent, 9-fluorenone-4-carbonyl chloride (FCC), is chosen to functionalize amino acids in alkaline medium before their HPLC resolution. FCC reacts with both primary and secondary amino acids to produce stable and highly fluorescent derivatives suitable for sensitive and efficient chromatographic determination and resolution on a teicoplanin chiral stationary phase (CSP) using the methanol-based solvent mixture as the mobile phase. The detection limit is in the picomole range and approximately 0.01% of the d-enantiomer in an excess of the l-enantiomer is detectable. However, the resolution is not reproducible under the elution of either the water- or the acetonitrile-based mobile phase. The increase in solubility of analyte in the mobile phase seems to be responsible. Upon comparison under the optimal chromatographic conditions, the resolution is better than that for the 9-fluorenylmethyl chloroformate (FMOC) or 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) derivatives reported previously.     

Twoplex 12/13C6 aniline stable isotope and linkage‐specific sialic acid labeling 2D‐LC‐MS workflow for quantitative N‐glycomics

Quantitative glycomics represents an actively expanding research field ranging from the discovery of disease‐associated glycan alterations to the quantitative characterization of N‐glycans on therapeutic proteins. Commonly used analytical platforms for comparative relative quantitation of complex glycan samples include MALDI‐TOF‐MS or chromatographic glycan profiling with subsequent data alignment and statistical evaluation. Limitations of such approaches include run‐to‐run technical variation and the potential introduction of subjectivity during data processing. Here, we introduce an offline 2D LC‐MS^E workflow for the fractionation and relative quantitation of twoplex isotopically labeled N‐linked oligosaccharides using neutral ¹²C₆ and ¹³C₆ aniline (Δmass = 6 Da). Additional linkage‐specific derivatization of sialic acids using 4‐(4,6‐dimethoxy‐1,3,5‐trizain‐2‐yl)‐4‐methylmorpholinium chloride offered simultaneous and advanced in‐depth structural characterization. The potential of the method was demonstrated for the differential analysis of structurally defined N‐glycans released from serum proteins of patients diagnosed with various stages of colorectal cancer. The described twoplex ¹²C₆/¹³C₆ aniline 2D LC‐MS platform is ideally suited for differential glycomic analysis of structurally complex N‐glycan pools due to combination and analysis of samples in a single LC‐MS injection and the associated minimization in technical variation.     

Quantitation of reduced and total glutathione at the femtomole level by high-performance liquid chromatography with fluorescence detection: application to red blood cells and cultured fibroblasts

A new rapid and highly sensitive HPLC method with ortho-phthalaldehyde (OPA) pre-column derivatization has been developed for determination of reduced glutathione (GSH) and total glutathione (GSHt) in human red blood cells and cultured fibroblasts. OPA derivatives are separated on a reversed-phase HPLC column with an acetonitrile–sodium acetate gradient system and detected fluorimetrically. An internal standard (glutathione ethyl ester) is added to facilitate quantitation. Total glutathione is determined after reduction of disulfide groups with dithiothreitol; the oxidized glutathione (GSSG) concentration is calculated by subtraction of the GSH level from the GSHt level. The assay shows high sensitivity (50 fmol per injection, the lowest reported), good precision (C.V. <5.0%), an analytical recovery of GSH and GSSG close to 100%, and linearity (r>0.999). This HPLC technique is very simple and rapid. Its wide applicability and high sensitivity make it a convenient and reliable method for glutathione determination in various biological samples.     

New procedure for isolation of amino acids based on selective hydrolysis of trimethylsilyl derivatives

A rapid procedure for the isolation of amino acids from physiological fluids by class separation suitable for gas chromatographic and gas chromatographic—mass spectrometric analysis is described. A physiological fluid such as plasma is adjusted to pH 2 and extracted with diethyl ether to remove organic acids and neutrals. After precipitation of proteins with trichloroacetic acid, the aqueous plasma is dried and derivatized by trimethylsilylation. Organic compounds like sugars and amino acids are rendered soluble in petroleum ether leaving inorganic salts when the soluble layer is transferred. Separation of sugars from amino acids is achieved by taking advantage of the different rates of aqueous hydrolysis of the trimethylsilyl (TMS) derivatives. Mixing the petroleum ether extract with a small volume of water results in two phases. The petroleum ether layer contains TMS-sugar constituents of plasma and the aqueous layer contains free amino acids and amines. This procedure was used to isolate L-dopa, 3-O-methyldopa and tyrosine from human plasma in a quantitation assay using ¹⁵O-labelled amino acids and gas chromatography—mass spectrometry.     

Selective photodestruction of α-amino acids

A problem typically encountered in the analysis of amino acids in chemical evolution experiments and in extracts of meteorites is the large number present. For example, α-, β-, and γ-amino acids, N-mono substituted α-amino acids, and dicarboxylic α-amino acids have been found in extracts of the Murchison meteorite, and many more amino acids are present than have been positively identified by computerized gas chromatographic mass spectrometry. This paper reports an analytical method to selectively destroy the α-amino acids, with only the β- and γ-amino acids remaining in the solution. It is based on the ability of Cu²⁺ to complex with amino acids, the order of stability of these complexes being α > β > γ, = δ, = ε = 0. Aqueous solutions of α-amino acid-Cu²⁺ chelates are known to be decomposed by 254 nm light as well as by nonmonochromatic uv light, yielding a precipitate of Cu₂O. This paper shows that at 254 nm (ligand-metal charge transfer band) the rate of destruction of amino acids in Cu²⁺ aqueous solutions is in the following order, dicarboxylic α-amino acids > α-amino acids > N-monosubstituted α-amino acids β-amino acids ≈ γ-amino acids. Thus by irradiation with 254 nm light in the presence of Cu²⁺ all the amino acids can be destroyed except the β- and γ-amino acids. When almost 100% of the α-amino acids are destroyed, 80% of the β- and γ-amino acids still exist in solution. With this procedure, complex mixtures of amino acids can be simplified to make identification by gas chromatographic mass spectrometry casier.     

A fast and sensitive method for measuring picomole levels of total free amino acids in very small amounts of biological tissues

Summary. In the present study we describe a simple and fast method to measure the concentration of total free amino acids in very small amounts of biological tissues. The procedure described here is based on the reaction of free amino acids with o-phthaldialdehyde (OPA) in the presence of a reducing agent, β-mercaptoethanol (MET), to give a complex which can be measured by fluorescence. It is a very rapid process and has the same reliability as the conventional ninhydrin method of Moore and Stein but is about 500 times more sensitive. The sensitivity of the new protocol is such to permit the determination with high reliability of very small amounts of free amino acids at picomole levels, either in a standard amino acid mixture or in biological tissues, without chromatographic separation of the amino acids. It is particularly useful when the amount of the sample is very low, e.g. on a single pituitary or pineal gland of small animals or on single cells, such as oocytes or eggs, as well as single ganglions or axons of marine invertebrates. Received September 22, 1999 Accepted July 5, 2000     

Evaluation of isotope discrimination in C-based metabolic flux analysis

In a ¹³C experiment for metabolic flux analysis (¹³C MFA), we examined isotope discrimination by measuring the labeling of glucose, amino acids, and hexose monophosphates via mass spectrometry. When Escherichia coli grew in a mix of 20% fully labeled and 80% naturally labeled glucose medium, the cell metabolism favored light isotopes and the measured isotopic ratios (δ¹³C) were in the range of −35 to −92. Glucose transporters might play an important role in such isotopic fractionation. Flux analysis showed that both isotopic discrimination and isotopic impurities in labeled substrates could affect the solution of ¹³C MFA.     

THE CELL-FREE SYNTHESIS OF ALKALOID-BINDING PROTEINS IN IMMATURE RAT BRAIN1

Abstract— cell-free amino acid incorporating system from immature rat brain, consisting of ribosomal and soluble fractions, has been investigated for its capacity to incorporate [¹⁴C]amino acids into specific soluble proteins that interact with vinblastine sulfate and colchicine. The soluble ¹⁴C-labeled proteins formed in the cell-free system during incubation were compared with similar soluble proteins from immature rat brain which had been labeled in vivo by the incorporation of ¹⁴C-labeled amino acids. Criteria for the formation of vinblastine-binding, ¹⁴C-labeled proteins were: (1) aggregation of ¹⁴C-labeled soluble protein by one mm -vinblastine sulfate and (2) immunoprecipitation of ¹⁴C-labeled soluble protein by an antiserum against vinblastine sulfate-precipitable material. Criteria for the formation of [³H]colchicine-binding, ¹⁴C-labeled protein were based upon: (1) co-precipitation of the ³H-and ¹⁴C-labeled materials by vinblastine sulfate and (2) the coincidence of ³H- and ¹⁴C-labeled elution peaks from columns of Sephadex G-200, DEAE-Sephadex A-50 and isoelectric focusing. Both in the in vitro and in the in vivo system, ¹⁴C-labeled amino acids were incorporated into soluble proteins of the post-microsomal supernatant fraction. Proteins labeled with ¹⁴C-labeled amino acids in vitro and in vivo yielded comparable and qualitatively identical results by the criteria tested, including the formation of immunoprecipitates. In the in vitro system, ¹⁴C-labeled amino acids were incorporated into protein with a molecular weight of approx 120,000, an isoelectric point of 5.3 and with a chromatographic mobility on Sephadex G-200 which is identical to [³H]colchicine-binding protein. The above experimental results are presumptive evidence for the synthesis of vinblastine-binding and colchicine-binding proteins in the in vitro cell-free system.     

反相高效液相色谱法测定烟叶中的游离氨基酸

用不同浓度的乙醇溶液提取烟样中的游离氨基酸 ,结果显示 ,存在最佳的乙醇溶液浓度 ,使烟样中被提取的游离氨基酸总量最大 ;对比了活性炭、乙醚、5 %磺基水杨酸、阳离子交换柱的纯化效果 ,发现阳离子交换柱的纯化效果较其它三种试剂要好。在提取和纯化之后 ,采用OPA、FMOC联合在线衍生反相高效液相色谱法测定了烟样中的游离氨基酸 ,该方法使烟样中的氨基酸和亚氨基酸能被同时测定 ,并且分析方法的重现性和回收率均令人满意。最后用该方法对云南B2 F98(上部、橘黄、二等烟叶 ,98年产 )烟叶中的游离氨基酸进行了测定 ,有 15种氨基酸被测出 ,其中Pro含量最高 ,约占总量的 2 5 % ,Thr含量最低 ,约占总量的 1%。     

High-performance liquid chromatographic determination of clindamycin in human plasma or serum: application to the bioequivalency study of clindamycin phosphate injections

This paper presents an assay of clindamycin phosphate injection in human plasma or serum. A 0.5-ml volume of plasma was used with the internal standard, propranolol. The sample was loaded onto a silica extraction column. The column was washed with deionized water and then eluted with methanol. The eluates were evaporated under nitrogen gas. The residue was reconstituted with the mobile phase and injected onto the high-performance liquid chromatographic system: a 5-μm, 25 cm×4.6 mm I.D. ODS2 column was used with acetonitrile, tetrahydrofuran and 0.05 M phosphate buffer as the mobile phase and with ultraviolet detection at 204 nm. A limit of quantitation of 0.05 μg/ml was found, with a coefficient of variation of 11.6% (n=6). The linear range is between 0.05 and 20.00 μg/ml and gives a coefficient of determination (r²) of 0.9992. The method has been successfully applied to the bioavailability study of two commercial preparations of clindamycin phosphate injection (300 mg each) in twelve healthy adult male volunteers.