Comparison of gas and liquid chromatography (GC, LC) coupled with isotope ratio mass spectrometry (IRMS): A case study on rice amino acids.

Organic nitrogen is now considered a significant source of N for plants. Although organic management practices increase soil organic C and N content, the importance of organic N as a source of crop N under organic farming management systems is still poorly understood. While dual-labeled (13C and 15N) molecule methods have been developed to study amino acid uptake by plants, multiple biases may arise from pre-uptake mineralization by microorganisms or post-uptake metabolism by the plant. We propose the combination of different isotopic analysis methods with molecule isotopologues as a novel approach to improve the accuracy of measured amino acid uptake rates in the total N budget of cucumber seedlings and provide a better characterization of post-uptake metabolism. Cucumber seedlings were exposed to solutions containing L-Ala-1-13C,15N or U-L-Ala-13C3,15N, in combination with ammonium nitrate, at total N concentrations ranging from 0 to 15 mM N and at inorganic/organic N ratios from 10:1 to 500:1. Roots and shoots were then subjected to bulk stable isotope analysis (BSIA) by Isotope Ratio Mass Spectrometry (IRMS), and to compound-specific stable isotope analysis (CSIA) of the free amino acids by Gas Chromatography – Combustion – Isotope Ratio Mass Spectrometry (GC-C-IRMS). Plants exposed to a lower inorganic:organic N ratio acquired up to 6.84% of their N from alanine, compared with 0.94% at higher ratio. No 13C from L-Ala-1-13C,15N was found in shoot tissues suggesting that post-uptake metabolism of Ala leads to the loss of the carboxyl-C as CO2. CSIA of the free amino acids in roots confirmed that intact Ala is indeed taken up by the roots, but that it is rapidly metabolized. C atoms other than from the carboxyl group and amino-N from Ala are assimilated in other amino acids, predominantly Glu, Gln, Asp, and Asn. Uptake rates reported by CSIA of the free amino acids are nevertheless much lower (16–64 times) than those reported by BSIA. Combining the use of isotopologues of amino acids with compound-specific isotope analysis helps reduce the bias in the assessment of organic N uptake and improves the understanding of organic N assimilation especially in the context of organic horticulture.

Adult blow flies are one of the first necrophagous insects to colonize fresh carcasses. The eggs they lay hatch into larvae, which then feed on the decomposing body. Like all organisms, blow flies "are what they eat," meaning that the isotopic composition of their body tissues reflects their diet. This manuscript combines ecology witha forensic application by using isotope ratio mass spectrometry (IRMS) to understand the relationship between the δ13C of amino acids in different carrion sources and the blow fly that feed on them. We also measure the amino acid-level fractionation that occurs at each major life stage of the blow flies. Adult blow flies from a commercial strain of Calliphora vicina (Robineau-Desvoidy) (Diptera: Calliphoridae) oviposited on raw pork muscle, beef muscle, or chicken liver. Larvae, pupae, and adult blow flies from each carrion were selected for amino acid compound-specific isotope analysis. Canonical discriminant analysis showed that flies were correctly classified to specific carrion types in 100% (original rules) and 96.8% (leave-one-out cross-validation [LOOCV]) of cases. Regarding life stages, we obtained 100% and 71% of correct classification in original rules and LOOCV, respectively. The isotope ratios of most of the essential amino acids did not significantly change between life stages (at 95% CI). However, some non-essential amino acids(Ala, Ser, and Glu) and some conditionally essential amino acids (Gly and Pro) were isotopically depleted in the adult stage. Except for the essential amino acids, the amino acids in larvae and pupae were enriched in 13C, and adult blow flies were depleted in 13C relative to the carrion on which they fed. These results make it possible to exclude potential sources of carrion as larval food. Amino acid-specific IRMS could help inform entomologists whether a fly has just arrived from another location to feed on a corpse or has emerged from a pupa whose feedstock was the corpse. Such insight could enhance the significance of blow flies for post-mortem interval determinations. The analytical ability to link organisms from one trophic level to another through the use of compound-specific isotope analysis of amino acids could have wide-reaching consequences in a variety of disciplines. Graphical abstract ᅟ.

Compound-specific stable carbon isotope analysis of amino acids by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) is a highly selective and sensitive method for probing the biosynthetic/diagenetic pathways, pool size and turnover rates of proteins, previously intractable to bulk isotope analyses. However, amino acids are polyfunctional, non-volatile compounds which require derivatisation prior to GC analysis. While a wide range of derivatives exist for the GC analysis of amino acids only a handful have been utilised for their GC/C/IRMS analysis. Significantly, none of those derivatives currently employed appear completely satisfactory and a thorough assessment of their relative utility is lacking. Seven derivatives (three previously reported and four novel) for obtaining delta(13)C values of amino acids via GC/C/IRMS analysis were compared. More specifically, standard mixtures of 15 protein amino acids were converted into N-acetylmethyl (NACME) esters, N-acetyl n-propyl (NANP) esters, N-acetyl i-propyl (NAIP) esters, N-trifluoroacetyl-i-propyl (TFA-IP) esters, N-pivaloyl methyl (NPME) esters, N-pivaloyl n-propyl (NPNP) esters and N-pivaloyl i-propyl (NPIP) esters. Each derivative was assessed with respect to its applicability to carbon isotope determinations of all the common alpha-amino acids, reaction yield, chromatographic resolution, stability, analyte-to-derivative carbon ratio, kinetic isotope effects and errors associated with their carbon isotope determinations. The NACME derivative was concluded to be the preferred derivative mainly due to the highest analyte-to-derivative carbon ratio being achieved, resulting in the lowest analytical errors for amino acid delta(13)C value determinations, ranging from +/-0.6 per thousand for phenylalanine, leucine and isoleucine to +/-1.1 per thousand for serine and glycine.

Results are presented of a comparison of the amino acid (AA) δ(13)C values obtained by gas chromatography-combustion-isotope ratio mass spectrometry (GC/C/IRMS) and liquid chromatography-isotope ratio mass spectrometry (LC/IRMS). Although the primary focus was the compound-specific stable carbon isotope analysis of bone collagen AAs, because of its growing application for palaeodietary and palaeoecological reconstruction, the results are relevant to any field where AA δ(13)C values are required. We compare LC/IRMS with the most up-to-date GC/C/IRMS method using N-acetyl methyl ester (NACME) AA derivatives. This comparison involves the analysis of standard AAs and hydrolysates of archaeological human bone collagen, which have been previously investigated as N-trifluoroacetyl isopropyl esters (TFA/IP). It was observed that, although GC/C/IRMS analyses required less sample, LC/IRMS permitted the analysis of a wider range of AAs, particularly those not amenable to GC analysis (e.g. arginine). Accordingly, reconstructed bulk δ(13)C values based on LC/IRMS-derived δ(13)C values were closer to the EA/IRMS-derived δ(13)C values than those based on GC/C/IRMS values. The analytical errors for LC/IRMS AA δ(13)C values were lower than GC/C/IRMS determinations. Inconsistencies in the δ(13)C values of the TFA/IP derivatives compared with the NACME- and LC/IRMS-derived δ(13)C values suggest inherent problems with the use of TFA/IP derivatives, resulting from: (i) inefficient sample combustion, and/or (ii) differences in the intra-molecular distribution of δ(13)C values between AAs, which are manifested by incomplete combustion. Close similarities between the NACME AA δ(13)C values and the LC/IRMS-derived δ(13)C values suggest that the TFA/IP derivatives should be abandoned for the natural abundance determinations of AA δ(13)C values.

To provide a simple one-step derivatization procedure for the analysis of a wide variety of amino acids in human hair by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA) derivatization is already widely used outside the IRMS community, is applicable to a variety of functional groups, and provides products that are common entries in mass spectral databases, thus simplifying compound identification. Method optimization and validation were performed on a mixture of ten standard amino acids found abundantly in human hair. The method was then applied to the analysis of scalp hair from six human subjects. The hair was washed, hydrolyzed with 6 M HCl, derivatized using BSTFA in acetonitrile and analyzed using gas chromatography (GC) with concurrent quadrupole and isotope ratio mass spectrometry (IRMS) detectors. The reproducibility for the δ(13)C measurements, including the derivatization procedure and GC/C/IRMS analysis, on a day-to-day comparison was between 0.19‰ and 0.35‰ (SD, N = 12), with an average standard deviation of 0.26‰. Because trimethylsilylation adds 3N carbon atoms (where N = # reactive protons) to each amino acid, the δ(13)C values for amino acid derivatives were corrected using a mass balance correction and the measured kinetic isotope effect (KIE). The KIE values ranged from 0.984 to 1.020. The procedure gave consistent δ(13)C values with precision similar to other derivatization methods for the range of sample sizes studied: 50-1000 µg of each amino acid. The method gave δ(13)C values consistent with the known literature values when applied to the analysis of amino acids in human hair.

Compound-specific stable isotope analysis of individual amino acids (AAs) has been widely used in studies on food webs, resource tracing, and biochemical cycling. In the present study, juvenile sea cucumbers Apostichopus japonicus were fed the microalga Cylindrotheca fusiformis (CF) or Sargassum thunbergii (ST) during a 130-day experiment. The δ13C values of individual AAs in the experimental diet and body wall of sea cucumbers were determined to calculate the variability in carbon isotopic fractionation (Δ13C) and elucidate the isotopic routing of essential AAs and biosynthesis of nonessential AAs. The results showed that the sea cucumbers fed with diet CF had higher specific growth and ingestion rates but relatively lower feed conversion efficiency compared to those fed with diet ST. The experimental diets were generally less abundant in nonessential AAs (i.e., glycine, serine, aspartic acid, and arginine) but more abundant in essential AAs (i.e., isoleucine, leucine, lysine, phenylalanine, and histidine) than body walls. The fluctuations in the δ13C values of total AAs analyzed were 19.8 ± 4.6‰ for diets and 21.3 ± 2.7‰ for body walls. Serine and threonine were 13C-enriched AAs, while leucine and phenylalanine were 13C-depleted AAs. The diet ST treatment exhibited more positive Δ13C values of nonessential AAs (e.g., glycine, alanine, aspartic acid, and proline) compared to diet CF. There were significant negative relationships between Δ13C values and differences in nonessential AA percent abundance between the experimental diets and body walls of sea cucumber (for diet CF: y = -0.79 - 0.56x, r2 = 0.47; diet ST: y = 0.75 - 0.29x, r2 = 0.51), which implied the flexibility in the routing of various dietary macronutrients (protein, lipids, and carbohydrates) by sea cucumber. This study can greatly provide a new understanding of nutrient utilization and metabolism routing during juvenile sea cucumber culturing.

The present study was conducted to examine the effect of different levels of essential and nonessential amino acid in NCSU-23 medium on the in vitro-produced porcine embryo as it develops from the zygote to the blastocyst stage. Four experiments were performed, each with a completely randomized design involving 5 to 8 replications of treatments. In order to know the effect of nonessential amino acids in NCSU-23 medium, 0, 5, 10 and 20 μl/ml MEM were supplemented there to, (Exp. 1) and the medium was supplemented with same level of essential amino acids (Exp. 2). The combined effect of nonessential (0, 5, 10 and 20 μl/ml MEM) and essential amino acids (0, 5, 10 and 10 μl/ml MEM) in NCSU-23 medium (Exp. 3), first 72 h with non-essential amino acids (at 0, 5, 10 and 20 μl/ml MEM), and last 4 d with essential amino acids with the same level as NEAA (Exp. 4) were examined. The embryo development was monitored and the quality of blastocysts was evaluated by counting the number of total cells and determining the ratio of inner cell mass (ICM) to trophoectoderm (TE) cells. When Eagle's nonessential amino acids (MEM) added to NCSU-23 medium, it significantly increased the likelihood of development to the 2- to 4-cell stage and subsequent blastocyst development. Supplementation of different levels of essential amino acids in the NCSU-23 medium decreased cleavage rate, rate of morula and blastocyst development and the number of ICMs. In the case of the combined effect of essential and nonessential amino acids, better and significant results were found for blastocysts, hatching blastocysts and for ICM numbers which were also dose dependent. With respect to the biphasic effect of nonessential and essential amino acids, nonessential amino acids increased cleavage whereas essential amino acids increased the total cell number. Neither the nonessential nor the essential group of amino acids, on their own, affected blastocyst cell number or the differentiation of cells in the blastocyst. In conclusion, this study determined the role of nonessential and essential amino acids in the culture of the porcine embryo and showed that the embryo requires different levels of amino acids as it develops from the zygote to the blastocyst stage.

Compound specific stable isotope analysis of aromatics in diesel fuel to identify potential cocktailing

Isotope ratio mass spectrometry (IRMS) measures small differences in the abundances of light-stable isotopes of elements, mostly carbon, hydrogen, oxygen, sulfur, and nitrogen. The ratio of isotopes is dependent on the environment where the samples are located and therefore the measurement of the isotope signature in food and environmental samples can give information to help to distinguish samples that share the same chemical composition but can be under different conditions. This chapter provides an overview of isotope ratio mass spectrometry (IRMS) in the context of food and environmental sample analysis (e.g., food authenticity, food origin, or environmental contamination). The fundamentals and latest developments of the technique, most important aspects of the instrumentation, analysis of isotope ratio information as well as key applications are described to illustrate the impact of this rapidly growing field of research especially in terms of the number and type of applications. Finally, applications in nutrition and trophic, environment, soil science, and food authenticity are assessed by giving details on the progresses, advantages, and pitfalls.KeywordsElemental analysisGas chromatographyIsotope ratio mass spectrometry (IRMS)Isotopic abundanceLiquid chromatographyMagnetic sector mass analyzer

Carbon isotope analysis by bulk elemental analysis coupled with isotope ratio mass spectrometry has been the mainstay of delta(13)C analyses both at natural abundance and in tracer studies. More recently, compound-specific isotope analysis (CSIA) has become established, whereby organic constituents are separated online by gas or liquid chromatography before oxidation and analysis of CO(2) for constituent delta(13)C. Theoretically, there should be concordance between bulk delta(13)C measurements and carbon-weighted delta(13)C measurements of carbon-containing constituents. To test the concordance between the bulk and CSIA, fish oil was chosen because the majority of carbon in fish oil is in the triacylglycerol form and approximately 95% of this carbon is amenable to CSIA in the form of fatty acids. Bulk isotope analysis was carried out on aliquots of oil extracted from 55 fish samples and delta(13)C values were obtained. Free fatty acids (FFAs) were produced from the oil samples by saponification and derivatised to fatty acid methyl esters (FAMEs) for CSIA by gas chromatography/combustion/isotope ratio mass spectrometry. A known amount of an internal standard (C15:0 FAME) was added to allow analyte quantitation. This internal standard was also isotopically calibrated in both its FFA (delta(13)C = -34.30 per thousand) and FAME (delta(13)C = -34.94 per thousand) form. This allowed reporting of FFA delta(13)C from measured FAME delta(13)C values. The bulk delta(13)C was reconstructed from CSIA data based on each FFA delta(13)C and the relative amount of CO(2) produced by each analyte. The measured bulk mean delta(13)C (SD) was -23.75 per thousand (1.57 per thousand) compared with the reconstructed bulk mean delta(13)C of -23.76 (1.44 per thousand) from CSIA and was not significantly different. Further analysis of the data by the Bland-Altman method did not show particular bias in the data relative to the magnitude of the measurement. Good agreement between the methods was observed with the mean difference between methods (range) of 0.01 per thousand (-1.50 to 1.30).

We report a novel method for the chromatographic separation and measurement of stable carbon isotope ratios (delta(13)C) of individual amino acids in hair proteins and bone collagen using the LC-IsoLink system, which interfaces liquid chromatography (LC) with isotope ratio mass spectrometry (IRMS). This paper provides baseline separation of 15 and 13 of the 18 amino acids in bone collagen and hair proteins, respectively. We also describe an approach to analysing small hair samples for compound-specific analysis of segmental hair sections. The LC/IRMS method is applied in a historical context by the delta(13)C analysis of hair proteins and bone collagen recovered from six individuals from Uummannaq in Greenland. The analysis of hair and bone amino acids from the same individual, compared for the first time in this study, is of importance in palaeodietary reconstruction. If hair proteins can be used as a proxy for bone collagen at the amino acid level, this validates compound-specific isotope studies using hair as a model for palaeodietary reconstruction. Our results suggest that a small offset observed in the bulk delta(13)C values of the hair and bone samples may be attributed to two factors: (i) amino acid compositional differences between hair and bone proteins, and (ii) differential turnover rates of the tissues and the amino acid pools contributing to their synthesis. This application proposes that hair may be a useful complementary or alternative source of compound-specific paleodietary information.

Resolving the bulk δ 15N values of ancient human and animal bone collagen via compound-specific nitrogen isotope analysis of constituent amino acids

Amino acids are the building blocks of proteins and the analysis of their (13)C abundances is greatly simplified by the use of liquid chromatography (LC) systems coupled with isotope ratio mass spectrometry (IRMS) compared with gas chromatography (GC)-based methods. To date, various cation exchange chromatography columns have been employed for amino acid separation. Here, we report strong anion exchange chromatography (SAX) coupled to IRMS with a Liquiface interface for amino acid δ(13)C determination. Mixtures of underivatised amino acids (0.1-0.5 mM) and hydrolysates of representative proteins (prawns and bovine serum albumin) were resolved by LC/IRMS using a SAX column and inorganic eluents. Background inorganic carbon content was minimised through careful preparation of alkaline reagents and use of a pre-injector on-line carbonate removal device. SAX chromatography completely resolved 11 of the 16 expected protein amino acids following acid hydrolysis in underivatised form. Basic and neutral amino acids were resolved with 35 mM NaOH in isocratic mode. Elution of the aromatic and acidic amino acids required a higher hydroxide concentration (180 mM) and a counterion (NO 3-, 5-25 mM). The total run time was 70 min. The average δ(13)C precision of baseline-resolved peaks was 0.75‰ (range 0.04 to 1.06‰). SAX is a viable alternative to cation chromatography, especially where analysis of basic amino acids is important. The technology shows promise for (13)C amino acid analysis in ecology, archaeology, forensic science, nutrition and protein metabolism.

The scope of compound-specific stable isotope analysis has recently been increased with the development of the LC IsoLink which interfaces high-performance liquid chromatography (HPLC) and isotope ratio mass spectrometry (IRMS) to provide online LC/IRMS. This enables isotopic measurement of non-volatile compounds previously not amenable to compound-specific analysis or requiring substantial modification for gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS), which results in reduced precision. Amino acids are an example of such compounds. We present a new chromatographic method for the HPLC separation of underivatized amino acids using an acidic, aqueous mobile phase in conjunction with a mixed-mode stationary phase that can be interfaced with the LC IsoLink for compound-specific delta13C analysis. The method utilizes a reversed-phase Primesep-A column with embedded, ionizable, functional groups providing the capability for ion-exchange and hydrophobic interactions. Baseline separation of 15 amino acids and their carbon isotope values are reported with an average standard deviation of 0.18 per thousand (n = 6). In addition delta13C values of 18 amino acids are determined from modern protein and archaeological bone collagen hydrolysates, demonstrating the potential of this method for compound-specific applications in a number of fields including metabolic, ecological and palaeodietary studies.

This study provides data for the effect of dietary non-essential amino acid composition on the delta(13)C values of individual amino acids in rainbow trout (Oncorhynchus mykiss) using liquid chromatography coupled to isotope ratio mass spectrometry (LC/IRMS). In this experiment, trout were reared either on a control diet or on three experimental diets, differing in the composition of non-essential/conditionally essential amino acids, for a period of 6 weeks. The control diet was a commercial trout starter feed with fish meal as the main protein source. The experimental diets contained no protein, only synthetic amino acids. Diet 1 resembled the composition of fish meal in both essential and non-essential amino acids, Diet 2 had all essential amino acids, but cysteine, glycine, proline and tyrosine were replaced by the corresponding amounts of their precursors, and in Diet 3 all non-essential amino acids were replaced by glutamate. LC/IRMS was used for the determination of delta(13)C values of individual amino acids from diets and tissues without derivatization. Diet affected the delta(13)C of individual amino acids in fish. For fish on Diets 1-3 amino acid delta(13)C values showed a similar trend: phenylalanine showed very little change from diet to body tissue. Arginine, lysine, tyrosine and proline showed strong depletion from diet to body tissue and glycine, alanine, aspartate and serine all showed variable but strong enrichment in (13)C. Improvements are necessary before all amino acid delta(13)C values can be determined; however, this study demonstrates that measuring amino acid isotopic signatures by LC/IRMS is a promising new technique for nutritional physiologists.